High-level vancomycin-resistant Staphylococcus aureus isolates associated with a polymicrobial biofilm

Abstract

Glycopeptides such as vancomycin are the treatment of choice for infections due to methicillin-resistant Staphylococcus aureus. This study describes the identification of high-level vancomycin-resistant S. aureus (VRSA) isolates in a polymicrobial biofilm within an indwelling nephrostomy tube in a patient in New York. S. aureus, Enterococcus faecalis, Enterococcus faecium, Micrococcus species, Morganella morganii, and Pseudomonas aeruginosa were isolated from the biofilm. For VRSA isolates, vancomycin MICs ranged from 32 to >128 microg/ml. VRSA isolates were also resistant to aminoglycosides, fluoroquinolones, macrolides, penicillin, and tetracycline but remained susceptible to chloramphenicol, linezolid, rifampin, and trimethoprim-sulfamethoxazole. The vanA gene was localized to a plasmid of approximately 100 kb in VRSA and E. faecium isolates from the biofilm. Plasmid analysis revealed that the VRSA isolate acquired the 100-kb E. faecium plasmid, which was then maintained without integration into the MRSA plasmid. The tetracycline resistance genes tet(U) and tet(S), not previously detected in S. aureus isolates, were identified in the VRSA isolates. Additional resistance elements in the VRSA isolate included a multiresistance gene cluster, ermB-aadE-sat4-aphA-3, msrA (macrolide efflux), and the bifunctional aminoglycoside resistance gene aac(6')-aph(2")-Ia. Multiple combinations of resistance genes among the various isolates of staphylococci and enterococci, including vanA, tet(S), and tet(U), illustrate the dynamic nature of gene acquisition and loss within and between bacterial species throughout the course of infection. The potential for interspecies transfer of antimicrobial resistance genes, including resistance to vancomycin, may be enhanced by the microenvironment of a biofilm.

FIG. 1.

PFGE profiles of SmaI restriction fragments of VRSA and MRSA genomic DNA. A vertical line separates VRSA (lanes 1 to 4) from MRSA (lanes 5 and 7 to 10). The fragment size (in kilobases) is indicated for the SmaI digest of the reference strain, S. aureus NCTC 8325 (lane 6) (std). Boxed areas indicate regions with variations in the restriction profiles. VRSA variants are designated A to D, and MRSA variants are designated E or F.

FIG. 2.

Plasmid content and Southern hybridization. (A) Uncut plasmids isolated from MRSA 2530, VRSA 595, and VRE (E. faecium 2547) were separated on a 0.8% agarose gel; the size standard shown is a supercoiled DNA (scDNA) ladder (size range, 2 to 16 kb; Invitrogen). The 25-kb size estimate is based on restriction fragment analysis of the MRSA 2530 plasmid; the 100-kb size is based on relative migration compared with the 120-kb plasmid from the Pennsylvania VRSA isolate and the 58-kb plasmid pLW1043 from the Michigan VRSA isolate (not shown). (B) Southern blot of A probed with a digoxigenin-labeled vanA gene fragment generated by PCR. (C) Southern blot to localize the tet(U) and tet(S) genes. Uncut plasmids from VRSA 595 (lane 1), MRSA 2530 (lane 2), and VRE 2547 (lane 3) were run in duplicate on the same gel and transferred to a Zeta-Probe membrane. The membrane was cut, and the two halves were hybridized with digoxigenin-labeled probes for either tet(U) or tet(S), as indicated.

FIG. 3.

Restriction analysis of VRSA, MRSA, and VRE plasmids from biofilm isolates. (A) BglII digests indicate a single restriction site in the MRSA plasmid. For VRSA 595, the additional fragments match those seen in the VRE plasmid. The “cured” VRSA is a vancomycin-susceptible derivative of VRSA 595. (B) EcoRI digests to compare MRSA 2530 plasmid fragments with those from the “cured” VRSA and VRSA 595.

FIG. 4.

Comparison of the vanA-containing genetic elements of pLW595 with the prototype Tn1546. ORF1 and ORF2 are represented by arrows filled with horizontal lines. Coding regions for van genes are shown as solid gray arrows. Triangles represent insertion sequences found in the Tn1546-like element from pLW595. Block arrows indicate the direction of transcription. Black arrowheads represent inverted repeat (IR) sequences.