Evaluation of Non-Tissue Culture- versus Tissue Culture-Treated Microplates for Oritavancin Susceptibility Testing

LETTER

Oritavancin is a recently approved lipoglycopeptide with activity against Gram-positive bacteria (1). For a number of reasons, oritavancin in vitro susceptibility testing has been a challenge in clinical microbiology laboratories (1, 2). Early oritavancin broth microdilution methods underestimated the potency of oritavancin by 16- to 32-fold, an issue that was addressed by incorporating polysorbate 80 (3). Oritavancin rapidly binds plastic microplate surfaces, but it was reported that 80 to 100% can be recovered by adding 0.002% polysorbate 80 (3). Following identification of this issue, Clinical and Laboratory Standards Institute (CLSI) oritavancin MIC quality control (QC) ranges were redefined under conditions incorporating 0.002% polysorbate 80 (4, 5). Other than polysorbate 80, incubation time, CO₂, pH, and Ca²⁺ ions have been evaluated for their effects on oritavancin MICs but have not been found to have a substantial impact in the presence of polysorbate 80 (5). However, effects of individual microplate types on oritavancin MICs have not been addressed. Neither the CLSI nor the European Committee on Antimicrobial Susceptibility Testing (EUCAST) mentions the use of tissue culture-treated microplates for bacterial susceptibility testing. However, the CLSI document Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically (M07-A10 [6]) does not actually specify the type of plastic microdilution tray to be used for microdilution antimicrobial susceptibility testing of bacteria (6), and the EUCAST document Method for the Determination of Broth Dilution Minimum Inhibitory Concentrations of Antifungal Agents for Yeasts states that tissue culture-treated microplates have been used to set most EUCAST MIC breakpoints for yeasts (7). Following an observation of challenges with oritavancin QC testing possibly linked to microplate type, we evaluated the effect of microplate types on oritavancin susceptibility testing, comparing oritavancin MICs generated using tissue culture-treated and non-tissue culture-treated microplates.

We studied a total of 126 clinical isolates recovered from 1996 to 2016 at the Mayo Clinic (Rochester, MN), including 20 methicillin-resistant Staphylococcus aureus (MRSA), 20 methicillin-susceptible S. aureus (MSSA), 20 methicillin-resistant Staphylococcus epidermidis (MRSE), 20 methicillin-susceptible S. epidermidis (MSSE), 20 vancomycin-susceptible Enterococcus faecalis, 6 vancomycin-resistant E. faecalis (3 with VanA, 3 with VanB), 7 vancomycin-susceptible Enterococcus faecium, and 13 vancomycin-resistant E. faecium (10 with VanA, 3 with VanB) strains. S. aureus ATCC 29213 and E. faecalis ATCC 29212 were used as quality control strains. Polystyrene tissue culture-treated microplates (catalog number 3799; Costar, Corning, NY) and polystyrene non-tissue culture-treated microplates (catalog number 3788; Costar) were studied. Oritavancin (Sigma-Aldrich, St. Louis, MO) solutions were freshly made, and 0.002% polysorbate 80 (Sigma-Aldrich) was maintained in oritavancin solutions throughout testing. Microplates containing serial 2-fold oritavancin dilutions were prepared by following CLSI guidelines (6). The same oritavancin dilutions and inoculum preparations were used for comparative microplate testing.

The oritavancin MIC of each QC strain was tested in quintuplicate with each microplate type. Tissue culture-treated microplate oritavancin MICs ranged from 1 to 2 μg/ml for S. aureus ATCC 29213 and from 0.25 to 0.5 μg/ml for E. faecalis ATCC 29212, out of acceptable QC ranges as defined by the CLSI (8). Conversely, when we used non-tissue culture-treated microplates, oritavancin MICs ranged from 0.015 to 0.06 μg/ml for ATCC 29213 and from 0.008 to 0.015 μg/ml for ATCC 29212, all of which were within the range for acceptability (8). Overall, for the clinical isolates, tissue culture-treated microplate oritavancin MIC₉₀ values were 4 doubling dilutions (i.e., 16-fold) higher than non-tissue culture-treated microplate oritavancin MIC₉₀ values for staphylococci and 2 to 6 doubling dilutions (i.e., 4- to 64-fold) higher for enterococci (Table 1). (Although some of these values would technically have been interpreted as nonsusceptible, they would not have been reported due to failed QC.)

TABLE 1.

Comparison of oritavancin MIC values as determined using tissue culture-treated and non-tissue culture-treated microplates^a

Organism (no. of isolates tested)	NTC microplate MIC (μg/ml)			TC microplate MIC (μg/ml)			TC to NTC MIC90 increase (doubling dilutions)
	Range	50%	90%	Range	50%	90%
Staphylococcus aureus
MRSA (20)	≤0.008–0.12	0.03	0.12	0.25–2	1	2	4
MSSA (20)	≤0.008–0.12	0.03	0.12	0.25–2	1	2	4
Staphylococcus epidermidis
MRSE (20)	≤0.008–0.5	0.03	0.12	0.06–2	1	2	4
MSSE (20)	≤0.008–0.5	0.015	0.12	0.25–8	1	2	4
Enterococcus faecalis
VSE (20)	≤0.002–0.06	≤0.002	0.015	0.06–2	0.5	1	6
VRE with VanA (3)	0.12–0.5	0.25	0.5	1–2	2	2	2
VRE with VanB (3)	0.004–0.3	0.015	0.03	0.5	0.5	0.5	4
Enterococcus faecium
VSE (7)	≤0.002	≤0.002	≤0.002	0.008–0.06	0.06	0.06	≥5
VRE with VanA (10)	0.03–0.25	0.06	0.12	0.5–1	1	1	3
VRE with VanB (3)	≤0.002	≤0.002	≤0.002	0.008–0.06	0.015	0.06	≥5

^aNTC, non-tissue culture-treated; TC, tissue culture-treated; MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; MRSE, methicillin-resistant S. epidermidis; MSSE, methicillin-susceptible S. epidermidis; VSE, vancomycin-susceptible enterococci; VRE, vancomycin-resistant enterococci.

We have demonstrated that addition of 0.002% polysorbate 80 to oritavancin does not effectively prevent the loss of oritavancin when tissue culture-treated microplates are used. Surfaces of tissue culture-treated microplates are modified to render them hydrophilic, facilitating cellular attachment. Previous studies that showed that polysorbate 80 as well as lysed horse blood prevents oritavancin binding and that polysorbate 80 prevents dalbavancin binding to plastic surfaces of microplates did not specify whether the microplates used were tissue culture treated or nontreated (3, 9). Whereas those studies focused on the use of supplements to prevent antimicrobial binding to plastic surfaces, this study has demonstrated that the type of microplate itself is also an important variable impacting oritavancin susceptibility testing; interestingly, it did not obviously affect dalbavancin MIC testing, at least vis-à-vis being in the QC range, in our previous studies (10, 11). It has been demonstrated that materials used in colistin susceptibility testing impact colistin MICs (12, 13), with non-tissue culture-treated colistin MICs being lower than tissue culture-treated colistin MICs (13). In the 2017 CLSI M100 document (8), the use of untreated microplates for oritavancin MIC testing is mentioned for the first time but only in the MIC troubleshooting guide; this guidance might be more helpful if it is also stated in a more noticeable location.

In conclusion, non-tissue culture-treated microplates should be used for oritavancin broth microdilution susceptibility testing. Guidelines for broth microdilution susceptibility should clearly specify whether tissue culture- or non-tissue culture-treated microplates (or either) are acceptable.