Multisite Detection of Tn1549-Mediated vanB Vancomycin Resistance in Multidrug-Resistant Enterococcus faecalis ST6 in Texas and Florida

ABSTRACT

In the United States, vanB-mediated resistance in enterococci is rare. We characterized three sequence type (ST) 6, vancomycin-resistant Enterococcus faecalis isolates causing bacteremia in unique patients in spatiotemporally distinct settings. Isolates were recovered between 2018 and 2020 in two cities in the United States (Houston, TX; Miami, FL). The isolates harbored the vanB operon on a chromosomally located Tn1549 transposon, and epidemiological data suggested multiple introductions of the vanB gene cluster into ST6 E. faecalis.

INTRODUCTION

The prevalence of vancomycin resistance in enterococci in the United States is overwhelmingly higher for Enterococcus faecium than Enterococcus faecalis (1). This phenotype is primarily mediated by vanA in the United States, with vanB remaining rare among enterococci. Indeed, the estimated prevalence of E. faecium (VREfm) and E. faecalis (VREfs) isolates harboring vanB in North America was around 3.6% and 1%, respectively (1997 to 2016) (2). In the United States, only sporadic reports of vanB resistance have been documented since, usually within a single institution (3, 4). Outside of the United States, vanB-mediated vancomycin resistance in enterococci is either endemic (e.g., Australia) (5) or exhibiting a recent sharp increase, as reported in some Western European countries (6, 7). Here, we describe the identification of ST6 E. faecalis isolates belonging to three unique patients with bacteremia in two U.S. states, each harboring the vanB operon on a Tn1549 transposon (also knowns as Tn5382-like or Tn1549/Tn5382 elements) in differing genomic contexts. When paired with epidemiological data, these findings suggest multiple introductions of the vanB gene cluster into ST6 E. faecalis.

Patients were identified through the Vancomycin-Resistant Enterococcal Bacteremia Outcomes Study (VENOUS), an ongoing global prospective study investigating the molecular and clinical epidemiology of enterococcal bloodstream infections (8). For inclusion in VENOUS, all eligible patients must have isolates from their first (index) positive blood culture available for further characterization. MICs to vancomycin for each index isolate were performed through Etest following the Clinical and Laboratory Standards Institute (CLSI) guidelines (9). Isolates were sequenced on Illumina and Oxford Nanopore Technologies platforms (supplementary material; Table S2). To test the transferability of the vanB gene cluster on Tn1549, a filter mating protocol for conjugative transfer was used for each of the three index E. faecalis isolates. Conjugation assay methods are described in the supplementary material. To identify potential extracellular circular intermediates of Tn1549, inverse PCR experiments with the three index E. faecalis isolates were performed (supplementary material).

We identified six E. faecalis isolates harboring vanB (Table 1) belonging to three unique patients in our 203-patient VENOUS cohort (1.5%). Patients were hospitalized in a major cancer center (Patient HTX1) or a general hospital (Patient HTX2) in Houston, TX (2018 and 2020, respectively), or in a Miami, FL, general hospital (Patient MFL1) in 2018. Patient HTX1 was undergoing active chemotherapy treatment for acute lymphocytic leukemia, but the remaining two patients were immunocompetent. Of these, two were directly admitted to the intensive care unit (ICU) due to either worsening kidney function or hypotension secondary to septic shock. While patients HTX1 and HTX2 had a history of hospitalization in the year prior to study enrollment, they were not admitted to the same hospitals during this time frame. The index positive blood culture from each patient was obtained on the first day of hospital admission. There was no documented history of recent international travel for any of the patients.

TABLE 1.

Patient demographic and hospitalization information^a

Characteristic	Patient HTX1	Patient MFL1	Patient HTX2
Institution	Houston, TX CC	Miami, FL HS	Houston, TX HS
Year of admission	2018	2020	2020
Length of stay, days	29	83	15
Age, yrs	40	79	51
Location prior to admission	Community/home	LTAC	Community/home
Location upon admission	Non-ICU	ICU	ICU
Antibiotic history 30 days prior to admission	FEP, LZD, MIN	FEP, MEM, VAN	None
History of hospitalization in past year?	Yes	No	Yes
No. of positive blood cultures during hospitalization	3	1	6b
Duration of bacteremia, days	3	3	5
Antibiotics given during hospitalization	AMP, CRO, LZD, MEM	LZD	SAM, DAP, GEN, MEM, VAN
Outcome	Death	Discharged	Discharged

^aCC, cancer center; HS, hospital system; LTAC, long-term acute care; FEP, cefepime; LZD, linezolid; MIN, minocycline; AMP, ampicillin; CRO, ceftriaxone; MEM, meropenem; VAN, vancomycin; DAP, daptomycin; SAM, ampicillin, sulbactam; GEN, gentamicin.

^b2/6 isolates from cultures were available for sequencing.

All isolates from patients’ follow-up blood cultures exhibited 3 to 12 single nucleotide polymorphisms (SNPs) relative to their index isolate (Table S1; Fig. 1), indicating that each patient was infected with the same strain throughout the course of bacteremia. Thus, only isolates from each patient's index blood culture (n = 3; isolates HTX1-1, HTX2-1, and MFL1) were further analyzed. The two Houston, TX, index isolates were more similar to each other than to MFL1, the latter differing by 181 whole-genome SNPs (Fig. S1). Comparison to E. faecalis V583, the first ST6 vancomycin-resistant E. faecalis reported in the United States, revealed wgSNP differences of >2000 SNPs (Fig. S2). All isolates harbored a chromosomally located Tn1549 conjugative transposon carrying the vanB operon (Fig. 1). All identified Tn1549 were ca. 33.8 kb and displayed 99% identity relative to a reference Tn1549 (10). In isolates HTX1-1 and HTX2-1, Tn1549 had nearly identical chromosomal insertion sites within genes encoding a bacterial sugar uptake phosphotransferase system (PTS) (10). In contrast, in E. faecalis MLF1, Tn1549 was inserted closer to the bacterial replication initiator gene dnaA. Interestingly, although the left and right ends and inverted right and left repeat regions of Tn1549 were identical in all three strains, in both E. faecalis HTX1-1 and HTX2-1, Tn1549 was flanked by additional mobile genetic elements (MGEs), specifically, insertion sequence IS256. However, these additional MGEs were not detected in MFL1. We did not detect conjugative transfer of Tn1549 from any of the three strains to the laboratory strain E. faecalis OG1RF under the chosen experimental conditions, nor were circular intermediates of Tn1549 observed with inverse PCR.

FIG 1.

Characterization of Tn1549 transposons harboring vanB. Characterization of transposon Tn1549 carrying the vanB operon in each index isolate (n = 3). Genes belonging to Tn1549 are depicted in aqua, genes comprising the vanB operon are shown in red, and mobile genetic elements are shown in orange. All other genes are depicted in purple.

Given the heterogeneous contexts of Tn1549, we next investigated structural variations in the chromosomes using long-read sequencing to further explore genetic relatedness among the strains. We confirmed that HTX1-1 and HTX2-1 possessed nearly identical chromosomal gene content and synteny (Fig. S3a), with a total size of roughly 3.11 Mb. However, the chromosome of MFL1 was significantly larger (ca. 3.28-Mb), and a 2.9-Mb inversion was noted relative to the Houston, TX isolates (Fig. S3b) within tRNA regions (Fig. S3c). Indeed, tRNA regions are known sites of genomic recombination and integration (11). All isolates were multidrug-resistant, possessing similar antimicrobial resistance (AMR) gene content, including the presence of aac(6’)-Ie-aph(2”)-Ia conferring high-level aminoglycoside resistance (Table S4). Isolates also possessed unique plasmid repertoires, ranging from 1 to 3 plasmids, and at least one plasmid associated with each index isolate harbored one or more AMR genes. (Fig. S4).

Tn1549 carrying vanB has been extensively observed in E. faecium clinical strains worldwide (12, 13) but far less in E. faecalis (14, 15). To our knowledge, only one other instance of Tn1549-mediated vanB vancomycin resistance in E. faecalis has been described in the United States, and the observation of additional IS256 MGEs flanking Tn1549 in the two Houston, TX isolates has not before been described in this context. We also identified two distinct chromosomal insertion points of Tn1549 in HTX1-1/HTX2-1 and MFL1, suggesting independent acquisitions of the vanB-carrying transposon in these isolates that is supported by strain geographical and temporal separation. Given that the chromosomal gene structure and synteny—including the context of insertion for Tn1549—in the two Houston, TX patients were nearly identical, these isolates may have originated from a common ancestor some time ago. However, the considerable SNP distance and the lack of a spatiotemporal link between patients provide evidence against a more recent direct transmission event between the two patients.

All vanB-carrying isolates belonged to ST6, a sequence type highly prevalent in clinical settings in the United States and known for enhanced virulence and multidrug-resistant phenotypes (8, 16). Our findings suggest that vanB-carrying Tn1549 might be circulating in U.S. enterococci and that a dearth of comprehensive U.S. enterococcal genomic surveillance has precluded further discovery of this resistance phenotype in E. faecalis.

In summary, we found three epidemiologically distinct instances of multidrug-resistant ST6 E. faecalis bacteremia isolates harboring a vanB-carrying Tn1549 transposon in the United States. This observation, along with the evolving emergence of multi-drug resistant E. faecalis, support the need for more extensive surveillance for these nosocomial pathogens.

Funding Statement

5T32AI055449-15 to Theresa M. Koehler/Michael Lorenz (T32 training grant)