Selection of Strains for Quality Assessment of the Disk Induction Method for Detection of Inducible Clindamycin Resistance in Staphylococci: a CLSI Collaborative Study

A nine-laboratory collaborative study was conducted to selectpositive and negative quality assessment control strains forthe detection of inducible clindamycin resistance in staphylococci.Four strains of Staphylococcus aureus were tested as unknownson 10 different days in each laboratory using the recently recommendedCLSI (formerly NCCLS) disk diffusion method and the inoculumpurity control method. Strains contained either macrolide-lincosamide-streptograminB (MLS_B) resistance genes encoded by erm(A) or erm(C) or a macrolideresistance efflux pump encoded by msr(A). Based upon the resultsof this study, strain UT 32 (now designated ATCC strain BAA-977)containing erm(A) is recommended as the positive control organismfor inducible clindamycin resistance. Strain UT 25 (now designatedATCC BAA-976), which harbors the efflux pump encoded by msr(A),is recommended as the negative control organism.

INTRODUCTION

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Introduction

Macrolide (e.g., erythromycin), lincosamide (e.g., clindamycin),and streptogramin (e.g., quinupristin-dalfopristin) antimicrobialagents (collectively MLS agents) are widely used in the treatmentof staphylococcal infections. Macrolide resistance may be dueto an active efflux mechanism encoded by msr(A) (conferringresistance to macrolides and type B streptogramins but not toclindamycin) or to a ribosomal target modification that affectsthe activities of macrolides, lincosamides, and type B streptogramins(MLS_B resistance). MLS_B resistance in staphylococci is usuallyencoded by erm(A) or erm(C) (2, 4, 5) and can be either constitutiveor inducible (MLS_Bi). While constitutive resistance to clindamycincan be detected by standard susceptibility testing methods,inducible clindamycin resistance is not detected by standardbroth- or agar-based susceptibility test methods (8, 10). Thus,standard susceptibility tests cannot reliably differentiatemsr(A) resistance (clindamycin susceptible) and MLS_Bi resistance(inducibly clindamycin resistant).

Fiebelkorn et al. recently described a practical disk diffusionmethod for the detection of MLS_Bi strains (D-zone test) (3).It involves placing standard erythromycin and clindamycin disksin adjacent positions from 15 to 26 mm apart on a Mueller-Hintonagar plate when performing the Clinical and Laboratory StandardsInstitute (CLSI; formerly the NCCLS) disk diffusion test. Inducibleresistance to clindamycin is manifested by a flattening or bluntingof the clindamycin zone of inhibition adjacent to the erythromycindisk, giving a D shape to the zone of inhibited growth. Thesame authors have also shown that the D-zone test can be performedby placing erythromycin and clindamycin disks 15 mm from edgeto edge in the heavy-inoculum area of standard blood agar platesused for purity checks with automated or broth-based susceptibilitytest systems (6). There is increasing interest in assessingthe incidence of inducible clindamycin resistance in hospitalsand the community (3, 9, 10). However, there are no standardizedquality assessment strains for training and competency assessmentpurposes for the D-zone test.

The purpose of this multicenter study was to evaluate and selectpositive and negative control strains for the detection of inducibleclindamycin resistance in Staphylococcus spp.

MATERIALS AND METHODS

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Materials and Methods

Study sites. The study was conducted during November 2003 in nine laboratories:Baylor University Medical Center, the Centers for Disease Controland Prevention, Duke University Medical Center, MassachusettsGeneral Hospital, Minnesota Department of Health Laboratory,National Institutes of Health Clinical Center, Stanford UniversityMedical Center, University of California at Los Angeles MedicalCenter, and University of Texas Health Science Center at SanAntonio. Four of the laboratories used a commercial broth microdilutioninstrument (MicroScan; Dade-Behring, Inc., West Sacramento,CA) or the VITEK or VITEK 2 instrument (bioMerieux, Inc., Durham,NC). Three of the laboratories routinely employed disk diffusiontesting of staphylococci, and two of the laboratories employedmore than one of the above methods for their testing.

Organisms. Four strains of Staphylococcus aureus were evaluated: one clindamycin-susceptiblestrain with msr(A) (labeled UT 25) and three strains with inducibleclindamycin resistance, including one strain with erm(A) (UT32) and two strains with erm(C) (UT 751 and UT 944). Stabilityof the resistance traits was assessed in two of the participatinglaboratories (Centers for Disease Control and Prevention andUniversity of Texas Health Science Center at San Antonio). Thisinvolved daily subcultures and performance of the D-zone teston each strain for 33 days.

Study design. Each of the four strains was tested on 10 different days bythree methods: the standard CLSI disk diffusion method withthe 15-µg erythromycin and 2-µg clindamycin disksplaced at 15 mm (designated NCCLS-15) and 26 mm (NCCLS-26) aparton Mueller-Hinton agar (7) and the inoculum purity control methodby placing the erythromycin and clindamycin disks on standardsheep blood agar plates 15 mm apart (purity-15) (6). For theinoculum purity control method, a standard 0.5 McFarland inoculumsuspension or a further dilution (e.g., 1:10 or 1:20 in salineaccording to the standard protocol for the broth-based susceptibilitytesting method used by the participating laboratory) was usedas the inoculum source. The two disks were placed in the initialinoculum area of expected confluent growth. Following 16 to18 h of incubation at 35°C in ambient air, all plates wereexamined for evidence of inducible clindamycin resistance (flatteningof the clindamycin zone adjacent to the erythromycin disk, ora "D-zone"). Any flattening of the clindamycin zone was considereda positive test reaction.

RESULTS AND DISCUSSION

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Results and Discussion

The results for the four test strains of S. aureus were recordedfor the nine participating laboratories. The expected resultswere negative D-zone tests for strain UT 25 and positive D-zonetests for strains UT 32, UT 751, and UT 944. A summary of theresults expressed as the percentage of correct results/totalnumber of tests is shown in Table 1. Seven of the nine participatinglaboratories observed the expected results with all tests performedwith the four test strains. One laboratory (center 8) erroneouslyinterpreted the NCCLS-26 results during the first 4 days oftesting but then did obtain the expected results on the final6 days of testing after a review of the results identified theerrors. The errors involved failure to note a flattening ofthe inner zone around the clindamycin disk when the strain hadthe appearance of a double zone of inhibition. When the zonewas examined carefully for any flattening in subsequent tests,all results were interpreted as positive. Three laboratoriesobserved the expected results with all tests performed withthe NCCLS-15 method but recorded that the results with two ormore organisms were subtle when the NCCLS-26 method was used.One laboratory (center 1) observed erroneous results with strainUT 25, with 2 of 10 results using both the NCCLS-15 method andthe NCCLS-26 method and 1 of 10 of the purity-15 tests; andwith strain UT 944, with 1 of 10 results using the NCCLS-26method. Center 1 also noted an inadequate inoculum density forthe purity plate method when attempting to use a commercialdirect inoculum wand (BBL Prompt System; Becton-Dickinson, Sparks,MD) to streak the purity plates prior to disk placement.

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TABLE 1. Summary of the D-zone test results

This study has assessed the potential utility of four strainsfor the purposes of training and competency assessment of laboratorystaff in the performance of macrolide disk induction testingfor detection of inducible clindamycin resistance in staphylococci.In fact, the results of this study illustrate the importanceof using such strains when testing is first initiated. Thoselaboratories with prior experience in D-zone testing generallyobtained very consistent results in accordance with the expectedfindings with each candidate strain. In contrast, some laboratorieshad difficulty in recognizing the more subtle D-zones when thedisks were placed 26 mm apart on Mueller-Hinton agar. The 26-mmdisk separation was proposed initially (3) because it providedthe convenience of simply placing the disks in adjacent positionsof a standard disk dispenser. Disk placement at a distance of15 mm provides a more obvious indication of a positive D-zonebut requires hand placement of the disks. None of the labs haddifficulty recognizing the D-zones obtained when the disks wereplaced 15 mm apart on either Mueller-Hinton or sheep blood agarplates. However, this study illustrated the importance of usingeither the 0.5 McFarland suspension directly or only a modestdilution of that suspension: i.e., no more than a 1:20 dilutionin order to avoid potential false-negative results. The BBLPrompt System wand should not be used to inoculate purity platesfor D-zone determinations.

The stability of clindamycin resistance in the three strainswith inducible resistance was assessed in two of the participatinglaboratories. A total of 32 tests were performed over a 33-dayperiod. Stable resistance was observed for all tests with UT32 [erm(A)]. Inducible clindamycin resistance was observed forthe first 16 tests with UT 751 [erm(C)]; however, this strainlost the inducible resistance and became clindamycin susceptiblein subsequent tests. Inducible clindamycin resistance was observedwith all tests with strain UT 944 [erm(C)], although the zoneof inhibition between erythromycin and clindamycin was lessdistinct than with UT 32. For these reasons, UT 32 was selectedas the best positive control strain for D-zone quality assessmenttesting. Following the study, UT 32 was deposited with the AmericanType Culture Collection (ATCC), and has received the cataloguenumber BAA-977. Strain UT 25 (now ATCC BAA-976) that containsmsr(A) performed acceptably in this study and can be recommendedas a D-zone negative control strain.

Failure to identify inducible clindamycin resistance may leadto clinical failure when clindamycin is used therapeutically(1, 10). On the other hand, the broad assumption of clindamycinresistance based on erythromycin resistance may prevent theuse of clindamycin in cases where it would be effective therapy.Since the occurrence of inducible resistance to clindamycinvaries widely by hospital and geographic region (9), it is importantto perform the D-zone test when staphylococci appear by routinetests to be erythromycin resistant and clindamycin susceptible.ATCC BAA-977 and ATCC BAA-976 have been accepted by the CLSIas the positive and negative control strains, respectively,for D-zone quality assessment purposes.

ACKNOWLEDGMENTS

We thank the technologists who participated in this study fortheir important contributions.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathology, The University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229. Phone: (210) 567-4088. Fax: (210) 567-2367. E-mail: jorgensen{at}uthscsa.edu.

REFERENCES

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