Clinical isolates Enterococcus gallinarum AIB39 and E. gallinarum GS1 were studied to establish whether the expression of vanC-1-mediated resistance may be inducible or constitutive. By growth curve analysis, strain AIB39 exhibited the same lag period (i.e., 1 to 1.5 h) whether it was subcultured to unsupplemented brain heart infusion broth or broth containing 6 g of vancomycin per ml, a growth pattern typical of constitutively expressed resistance. Use of high-performance liquid chromatography ( Acquired vancomycin resistance among enterococci, most usually associated with Enterococcus faecium and Enterococcus faecalis, is mediated by vanA and vanB genes that encode ligases capable of producing altered UDP-MurNAc-pentapeptide precursors for which vancomycin has diminished affinity (1). The vancomycin resistance exhibited by Enterococcus gallinarum is generally considered a constitutively expressed intrinsic characteristic of this species (1,18,27,28). The intrinsic nature of this resistance is supported by the finding that the vanC-1 gene, which encodes a ligase that catalyzes the formation of D-Ala-D-Ser, has consistently been found in every E. gallinarum strain examined and has not been found in other enterococcal species (9,18,22). However, recent findings suggest that although resistance may be intrinsic, expression may not always be constitutive (3,22).For other gram-positive organisms considered to be intrinsically and constitutively resistant, such as Leuconostoc, Lactobacillus, and Pediococcus species, evidence suggests the presence of a single ligase system (3, 15). The only peptidoglycan precursor found in these organisms is UDP-In contrast, recent reports have established the likely existence of at least two ligases in E. gallinarum. This organism has exhibited the capacity to produce UDP-MurNAc-L-Ala-DGlu-L-Lys-D-Ala-D-Ala (UDP-MurNac-tetrapeptide-D-Ala) cell wall precursor and the altered precursor MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ser (UDP-MurNac-tetrapeptide-D-Ser), which is catalyzed by the vanC-1-encoded ligase (3, 22). The ability to produce the wild-type cell wall precursor UDPMurNac-tetrapeptide-D-Ala and one with a decreased affinity for vancomycin (i.e., UDP-MurNac-tetrapeptide-D-Ser) is a feature that is strikingly similar to the inducible, acquired glycopeptide resistance encoded by vanA and vanB. In these systems induction results in the production of UDP-MurNactetrapeptide-D-Lac that has significantly reduced vancomycin affinity compared with that of the UDP-MurNac-tetrapeptide-D-Ala peptidoglycan precursor (1, 4, 10).The presence of two ligases in E. gallinarum, a species heretofore considered to express resistance constitutively, is somewhat perplexing. In this report we establish the ability of E. gallinarum to inducibly or constitutively express vanC-1-mediated vancomycin resistance. Although vanC-1 may be intrinsic to this species, these findings raise several questions regarding the intrinsic nature of the regulatory mechanisms that accompany vanC-1. MATERIALS AND METHODSSource, i...

Surveillance cultures for vancomycin-resistant enterococci (VRE) and subsequent characterization of the isolates can be extremely burdensome and difficult. Therefore, efficient and reliable schemes for the characterization of surveillance isolates are needed. In this study, a commercial agar (bile esculin azide agar with 6 g of vancomycin per ml [BEAA]; Remel, Lenexa, Kans.) was used in the initial screening step to establish relatively rapid (i.e., in <24 h from the time of isolation) phenotype-based and PCR-based schemes for the detection and characterization of VRE. The phenotype-based scheme included Gram staining of growth on BEAA and subculture of cocci on sheep blood agar plates for vancomycin disk diffusion and pyrazinamidase (PYR) testing. For the PCR scheme, inocula for van gene detection were taken directly from the BEAA plates. The phenotypic approach was applied to 378 surveillance cultures that yielded growth on BEAA. Gram staining quickly eliminated gram-positive bacilli from further testing, and a negative PYR test classified 25 additional isolates as probable pediococci. A positive PYR test reliably identified 121 single-patient VRE isolates that included 83 Enterococcus faecium, 33 E. gallinarum, and 5 E. casseliflavus strains. The vancomycin inhibition zone size clearly distinguished VanA and VanB strains from VanC strains within 24 h of BEAA isolation. All VanA and VanB strains failed to produce zones of >6 mm, while only one VanC strain produced a zone of <15 mm. Challenging this phenotypic scheme with 47 stock VRE isolates produced similar findings. In direct PCR analyses, false-negative vanA and vanB results occurred with 12% of the specimens. Many of the false-negative reactions also failed to produce an internal control product, which underscores the need for including control primers when a PCR scheme is used. In summary, the phenotype-and the PCR-based schemes provide efficient methods for characterizing VRE within 24 h of isolation.

We investigated the accuracy of the recently released Remel Synergy Quad plate, a commercially available agar screening method for detecting high-level aminoglycoside and vancomycin resistance among enterococci that is based on the National Committee for Clinical Laboratory Standards recommended guidelines (National Committee for Clinical Laboratory Standards, M7-A3, 1993). The Synergy Quad correctly determined the gentamicin and streptomycin resistance status for Ն97% of 147 Enterococcus faecalis and Enterococcus faecium isolates tested. Detection of vancomycin resistance also was reliable, as no false susceptibility occurred with 36 vancomycin-resistant E. faecalis and E. faecium strains and false resistance occurred only once with the 47 susceptible strains tested. One strain each of Enterococcus gallinarum and Enterococcus casseliflavus failed to grow on the screen, but because the true nature and significance of resistance in such isolates is unknown the implication of their screen negativity is uncertain. In summary, the Remel Synergy Quad provides a highly accurate and convenient method for susceptibility testing of enterococci against gentamicin, streptomycin, and vancomycin.

Studies were conducted to identify factors contributing to the inability of the Vitek Gram-Positive Susceptibility system (GPS; bioMerieux, Vitek, Inc., Hazelwood, Mo.) to reliably detect vanB-mediated vancomycin resistance among enterococci. To some extent the accuracy of the GPS depended on a particular strain's level of resistance, as all isolates for which vancomycin MICs were >128 g/ml were readily detected but detection of resistance expressed by several strains for which MICs were <64 g/ml was sporadic. Factors besides the level of resistance were studied in two vanB strains. For one strain (Enterococcus faecium U8304), the ability of GPS to detect resistance was accurate and consistent, while for the other (Enterococcus faecalis V583), GPS results were inconsistent and unreliable. Using these isolates, we established that growth medium had the most notable effect on the detection of resistance. In the absence of vancomycin, Vitek GPS broth supported growth comparable to that obtained with brain heart infusion broth for both E. faecium U8304 and E. faecalis V583. However, in the presence of vancomycin the growth patterns changed dramatically so that neither VanB strain grew well in Vitek broth, and growth of V583 was barely detectable after 8 h of incubation. In contrast, good growth of both strains was observed in brain heart infusion broth supplemented with vancomycin. Additionally, the same medium effect was observed with other inducibly resistant VanB strains. In conclusion, although Vitek broth can support good enterococcal growth, this medium does not sufficiently support expression of vancomycin resistance by certain strains to allow them to be detected by the Vitek automated system. Furthermore, this observation establishes that the type of growth medium used can substantially influence the expression of vancomycin resistance and indicates that medium-based strategies should be explored for the enhancement of resistance detection among commercial systems.