Research Article
1 October 2001

Evaluation of the Vitek Card GPS105 and VTK-RO7.01 Software for Detection of Oxacillin Resistance in Clinically Relevant Coagulase-Negative Staphylococci

ABSTRACT

The performance of Vitek cards GPS105 with software version VTK-R07.01 for detection of oxacillin resistance in coagulase-negative staphylococci (CoNS) was compared to disk diffusion and PCR detection for mecA. The sensitivity and specificity of the Vitek GPS105 method were 97.6 and 85.5%, respectively.
Coagulase-negative staphylococci (CoNS) are important causes of nosocomial bacteremia, especially in patients with intravenous catheters or indwelling medical devices (1, 3, 7, 14). In some institutions, 70 to 75% of CoNS are resistant to oxacillin; consequently, vancomycin is the therapy of choice. If the isolate is oxacillin susceptible, treatment with a penicillinase-resistant penicillin is preferred to minimize selection of vancomycin-resistant organisms. A susceptibility test method that provides accurate oxacillin results is critical.
The National Committee for Clinical Laboratory Standards (NCCLS) changed the breakpoints for oxacillin testing of CoNS in January 1999, because of discrepancies between results of mecA genetic assays, which are considered the reference method for detecting oxacillin resistance in staphylococci, and the former NCCLS breakpoints of ≥4 μg/ml for resistance and ≤2 μg/ml for susceptibility. Currently, for microtiter dilution and disk diffusion testing, respectively, CoNS isolates with an MIC of ≥0.5 μg/ml or a zone diameter of ≤17 mm are considered resistant, and those with an MIC of ≤0.25 μg/ml or a zone diameter of ≥18 mm are considered susceptible (9). To comply with these guidelines, it was necessary for laboratories using the Vitek (bioMerieux Vitek, Inc.) to use an alternate method when testing CoNS. Vitek updated its software and increased the number of oxacillin concentrations tested to accommodate the revised oxacillin breakpoints for CoNS in September 1999. The purpose of the present study was to evaluate the performance of the Vitek card GPS105 with software version VTK-R07.01. The results were compared to disk diffusion and detection of the mecA gene by PCR.
Isolates of CoNS (n = 202) from cultures of clinical specimens of blood and urine were collected prospectively and stored at −70°C. Prior to testing, isolates were subcultured twice on sheep blood agar. Isolates were identified as CoNS using Gram stain, catalase, and latex agglutination tests (Staphaurex; Murex Biotech Ltd., Dartford, England). CoNS were identified to species with the API Staph-Ident system (bioMerieux Vitek, Inc.). Disk diffusion testing was performed according to NCCLS guidelines (10) by using Mueller-Hinton agar without additional NaCl (BD Biosciences, Sparks, Md.) and 1-μg oxacillin disks (BD Biosciences). Zone diameters were measured after plates were incubated in ambient air at 35°C for 24 h. Vitek cards GPS105 were inoculated and incubated according to manufacturer's recommendations. Results were interpreted by Vitek software version VTK-R07.01. If disk diffusion and Vitek results disagreed, both tests were repeated. Multiplex PCR for detection of themecA gene and the Staphylococcus IS431 sequence was performed as previously described (13); if isolates did not contain the mecA gene or an IS431 sequence, amplification of the Staphylococcus 16S rRNA sequence was performed as described previously (6).
The 202 CoNS isolates tested (141 from urine, 61 from blood) in this study belonged to 11 species (Table 1). The mecA gene was detected in 126 isolates (62.4%), of five species: Staphylococcus epidermidis, S. haemolyticus, S. hominis, S. warneri, and S. xylosus. S. capitis,S. cohnii, S. lugdunensis, S. saprophyticus, S. sciuri, and S. simulanswere consistently mecA negative.
Table 1.
Table 1. Vitek and disk diffusion oxacillin results after initial testing
Sourcea and speciesmecAresultTotal no.Disk diffusion (no. of isolates)No. of isolates with Vitek result (MIC [μg/ml]) of:
SensitiveResistant≤0.250.524≥8
Blood         
S. epidermidis11110110000
S. epidermidis+39039014727
S. hominis22020000
S. hominis+30300102
S. haemolyticus11010000
S. haemolyticus+20201001
S. lugdunensis11010000
S. warneri+20201010
Urine         
S. epidermidis34340330001
S. epidermidis+71269276947
S. hominis55050000
S. hominis+51431001
S. haemolyticus44040000
S. haemolyticus+30311100
S. capitis43131000
S. cohnii10110000
S. lugdunensis21102000
S. saprophyticus61506000
S. sciuri21111000
S. simulans22020000
S. warneri11010000
S. xylosus+10100001
a
A total of 61 CoNS isolates from blood and 141 from urine specimens were tested.
Initial Vitek and disk diffusion results are summarized by source in Table 1. The majority of the CoNS from blood were S. epidermidis. The mecA gene was detected in 44 (72%) of all blood isolates; all were determined to be oxacillin resistant by both disk diffusion and Vitek. All 115 mecA-negative isolates were determined to be susceptible by Vitek and disk diffusion (sensitivity and specificity of 100%).
Of the urine isolates (n = 141), 80 weremecA positive and 61 were mecA negative. Both Vitek and disk diffusion methods did not perform as well on isolates of CoNS from urine: 6 of the mecA positive isolates were determined to be susceptible by Vitek, and 11 of the mecAnegative isolates were determined to be resistant (sensitivity, 92.5%; specificity, 82%). The 11 isolates that were mecA negative but oxacillin resistant as determined by Vitek and disk diffusion included six S. saprophyticus isolates, two S. lugdunensis isolates, and one isolate each of S. capitis, S. cohnii, and S. sciuri. Three species (S. epidermidis, S. hominis, and S. haemolyticus) comprised the majority of the isolates (122 of 141; 86.5%). For these three species, the sensitivity and specificity of Vitek were high (92.4 and 97.6%, respectively). After repeat testing, Vitek correctly identified 76 of the 79 mecA-positive isolates as resistant and 100% of the mecA-negative isolates as susceptible (sensitivity, 96.2; specificity, 100%).
Discrepancies between results of mecA PCR assays when CoNS are tested and the NCCLS microtiter dilution and disk diffusion breakpoints for oxacillin against S. aureus have been recognized for several years. For this reason, various investigators recommended lowering the oxacillin MIC breakpoint for CoNS: York et al. (15) suggested ≤1 μg/ml for susceptibility, Cormican et al. (2) and McDonald et al. (8) suggested ≤0.5 μg/ml, and Marshall et al. (7) suggested ≤0.25 μg/ml.
Tenover et al. (12) concurred with the breakpoints of ≤0.25 μg/ml for susceptibility and ≥0.5 μg/ml for resistance because these values allowed maximum sensitivity for detectingmecA-positive isolates of S. epidermidis without drastically compromising specificity. These authors acknowledged, however, that these breakpoints were considerably less specific formecA in species of CoNS other than S. epidermidis. Tenover et al. (12) also suggested modified breakpoints for disk diffusion: ≤17 mm for resistance and ≥18 mm for susceptibility. The NCCLS considered data from all studies and adopted the oxacillin MIC and disk diffusion breakpoints for CoNS advocated by Tenover et al. Our results show that the new Vitek revised cards and software system accurately predicts the presence or absence of mecA in the three most commonly encountered species of CoNS.
For species of CoNS other than S. epidermidis, S. hominis, and S. haemolyticus, we found poor correlation between mecA PCR and oxacillin testing by Vitek or disk diffusion. In particular, isolates of S. capitis, S. cohnii, S. lugdunensis, S. saprophyticus, and S. sciuri that had an oxacillin MIC of 0.5 μg/ml as determined by Vitek (and were resistant by disk diffusion) were consistently mecA negative. Similar findings have been reported by other investigators (4, 5, 12). Hussain et al. (4) tested 493 isolates of 11 species of CoNS (no S. scuiri) and found mecA-negative isolates of S. capitis, S. cohnii, S. lugdunensis, and S. saprophyticus with oxacillin MICs of 0.5 to 2.0 μg/ml by agar dilution. In a second study, Hussain et al. (5) tested 463 isolates of 13 species of CoNS (no S. scuiri) and foundmecA-negative S. capitis, S. cohnii, S. lugdunensis, S. saprophyticus, S. caprae, S. warneri, andS. xylosus with oxacillin MICs of ≥0.5 μg/ml. In the latter study, isolates were tested by a latex agglutination test that detects penicillin-binding protein (PBP) 2a. Compared tomecA PCR, the sensitivity of that latex test was 100% and the specificity was 99.5%, whereas the specificity of the new NCCLS breakpoint was 60.8%. The mechanism(s) of decreased susceptibility to oxacillin in mecA-negative CoNS with an oxacillin MIC of 0.5 to 2.0 μg/ml is unknown, but alterations in PBPs other than PBP2a have been documented in some strains of S. haemolyticus andS. saprophyticus (11) and, therefore, suggested as one possibility (12). It is not known whether penicillinase-resistant penicillins can eradicate clinical isolates ofmecA-negative CoNS with an oxacillin MIC of 0.5 to 2.0 μg/ml. Clinical trials designed to answer this question are needed.

Acknowledgments

We thank Promega Corporation (Madison, Wis.) for providing the PCR reagents used in this study. BioMerieux kindly provided the API Staph Identification kits.

REFERENCES

1.
Cockerill F. R. III, Hughes J. G., Vetter E. A., Meuller R. A., Weaver A. L., Ilstrup D. M., Rosenblatt J. E., and Wilson W. R. Analysis of 281,797 consecutive blood cultures performed over an eight-year period: trends in microorganisms isolated and the value of anaerobic culture of blood.Clin. Infect. Dis.241997403-418
2.
Cormican M. G., Wile W. W., Barrett M. S., Pfaller M. A., and Jones R. N. Phenotypic detection of mecA-positive staphylococcal blood stream isolates: high accuracy of simple disk diffusion tests.Diagn. Microbiol. Infect. Dis.251996107-112
3.
Emori T. G. and Gaynes R. P. An overview of nosocomial infections, including the role of the microbiology laboratory.Clin. Microbiol. Rev.61993428-442
4.
Hussain Z., Stoakes L., Massey V., Diagre D., Fitzgerald V., El Sayed S., and Lannigan R. Correlation of oxacillin MIC with mecA gene carriage in coagulase-negative staphylococci.J. Clin. Microbiol.382000752-754
5.
Hussain Z., Stoakes L., Garrow S., Longo S., Fitzgerald V., El Sayed S., and Lannigan R. Rapid detection of mecA-positive and mecA-negative coagulase-negative staphylococci by an anti-penicillin binding protein 2a slide latex agglutination test.J. Clin. Microbiol.3820002051-2054
6.
Jaffe R. I., Lane J. D., Albury S. V., and Niemeyer D. M. Rapid extraction from and direct identification in clinical samples of methicillin-resistant staphylococci using the PCR.J. Clin. Microbiol.3820003407-3412
7.
Marshall S. A., Wilke W. W., Pfaller M. A., and Jones R. N. Staphylococcus aureus and coagulase-negative staphylococci from blood stream infections: frequency of occurrence, antimicrobial susceptibility, and molecular (mecA) characterization of oxacillin resistance in the SCOPE Program.Diagn. Microbiol. Infect. Dis.301998205-214
8.
McDonald C. L., Maher W. E., and Fass R. J. Revised interpretation of oxacillin MICs for Staphylococcus epidermidis based on mecA detection.Antimicrob. Agents Chemother.391995982-984
9.
National Committee for Clinical Laboratory Standards Performance standards for antimicrobial susceptibility testing, 11th information supplement: M100-211 21 2001, no. 1. National Committee for Clinical Laboratory Standards, Wayne, Pa.
10.
National Committee for Clinical Laboratory Standards Performance standards for antimicrobial disk susceptibility tests 7th ed. 2000 Approved Standard M2–A7. National Committee for Clinical Laboratory Standards, Wayne, Pa.
11.
Suzuki E., Hiramatsu K., and Yokota T. Survey of methicillin-resistant clinical strains of coagulase-negative staphylococci for mecA gene distribution.Antimicrob. Agents Chemother.361992429-434
12.
Tenover F. C., Jones R. N., Swenson J. M., Zimmer B., McAllister S., and Jorgensen J. H. for the NCCLS Staphylococcal Working Group Methods for improved detection of oxacillin resistance in coagulase-negative staphylococci: results of a multicenter study.J. Clin. Microbiol.3719994051-4058
13.
Vannuffel P., Gigi J., Ezzedine H., Vandercam B., Delmee M., Wauters G., and Gala J. L. Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR.J. Clin. Microbiol.3319952864-2867
14.
Weinstein M. P., Towns M. L., Quartey S. M., Mirrett S., Reimer L. G., Parmigiani G., and Reller L. B. The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults.Clin. Infect. Dis.241997584-602
15.
York M. K., Gibbs L., Chehab F., and Brooks G. F. Comparison of PCR detection of mecA with standard susceptibility testing methods to determine methicillin resistance in coagulase-negative staphylococci.J. Clin. Microbiol.341996249-253

Information & Contributors

Information

Published In

cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 39Number 101 October 2001
Pages: 3733 - 3735
PubMed: 11574604

History

Received: 28 March 2001
Returned for modification: 16 May 2001
Accepted: 11 July 2001
Published online: 1 October 2001

Permissions

Request permissions for this article.

Contributors

Authors

Fernando Martinez
Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0740
Laura J. Chandler
Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0740
Barbara S. Reisner
Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0740
Gail L. Woods
Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0740

Metrics & Citations

Metrics

Note:

  • For recently published articles, the TOTAL download count will appear as zero until a new month starts.
  • There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.
  • Citation counts come from the Crossref Cited by service.

Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. For an editable text file, please select Medlars format which will download as a .txt file. Simply select your manager software from the list below and click Download.

View Options

Figures and Media

Figures

Media

Tables

Share

Share

Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy