Evaluation of the accuracy of various phenotypic tests to detect oxacillin resistance in coagulase-negative staphylococci

ORIGINAL PAPERS

Evaluation of the accuracy of various phenotypic tests to detect oxacillin resistance in coagulase-negative staphylococci

Leandro Reus Rodrigues Perez^{I, II}; Pedro Alves d'Azevedo^III

^IMicrobiology Unit, Mãe de Deus Hospital

^IIPostgraduate Program in Pharmaceutical Sciences, Federal University of Rio Grande do Sul (UFRGS)

^IIIPostgraduate Program in Medical Sciences, Federal School Foundation of Medical Sciences of Porto Alegre (FFFCMPA); Porto Alegre, RS, Brazil

^{Address for correspondence}

ABSTRACT

In this study, we determined the accuracy of phenotypic tests (cefoxitin agar dilution, 30µg-cefoxitin and 1µg-oxacillin disks) to detect the oxacillin resistance/mecA gene among coagulase-negative staphylococci (CoNS) isolates. The presence of the mecA gene was detected by PCR technique (gold standard). A total of 176 CoNS isolates from blood of hospitalized patients were evaluated. Of these, 138 (78.4%) harbored the mecA gene. Using 30µg-cefoxitin and 1µg-oxacillin disks we obtained 100 and 98.3% accuracy, respectively. In addition, when cefoxitin was used as marker in an agar dilution method, the higher accuracy (99.4%) was established with 8mg cefoxitin per mL breakpoint. Thus, despite of the agar dilution method using cefoxitin as a marker not being standard for this detection, our results suggested that it is an excellent alternative to detect the oxacillin resistance/mecA gene among CoNS isolates.

Key-Words: Oxacillin, cefoxitin, CoNS.

Coagulase-negative staphylococci (CoNS), as a group, are a frequently isolated microorganism in nosocomial infections. In Brazil, about 10%-20% of bloodstream infections are caused by CoNS and, of the isolates, about 70%-90% present resistance to oxacillin [1].

Oxacillin resistance in CoNS is due to the acquisition of the mecA gene that encodes an additional protein, called PBP 2', with low affinity to the β-lactamic antimicrobials [2,3]. The accurate determination of oxacillin resistance is necessary for the establishment of therapeutic and surveillance programs [4]. The use of 30µg-cefoxitin disks has been established by the Clinical and Laboratory Standard Institute (CLSI, formerly NCCLS). However, no dilution method using cefoxitin as a marker for oxacillin resistance characterization has been adopted. Thus, the aim of this study was to evaluate the accuracy of phenotypic methods (cefoxitin agar dilution, 30µg-cefoxitin and 1µg-oxacillin disks by disk-diffusion) in detecting oxacillin resistance among CoNS isolates and to compare the results with the presence of the mecA gene detected by PCR (gold standard).

Materials and Methods

Bacterial Strains and Identification

From August to December 2004, 176 consecutive CoNS isolates were obtained from blood cultures of patients admitted to three hospitals: Hospital de Clínicas, Irmandade Santa Casa de Misericórdia and Hospital Conceição, in Porto Alegre, RS, Brazil. Only one isolate of each patient was included in the study. The isolates were identified to the species level as previously described [5]. All samples were frozen and stored at -20ºC in skim milk with 10% glycerol.

Antimicrobial Susceptibilities Testing

The susceptibilities to cefoxitin and oxacillin were determined by the disk diffusion method on Mueller-Hinton agar (Oxoid, Basingstoke, UK) plates using bacterial suspensions with turbidity adjusted for 0.5 McFarland standard (Densimat, Rome, Italy), after incubation at 35 ºC for 24h. The results were interpreted according to the CLSI (2006) guidelines.

The minimum inhibitory concentrations (MICs) for cefoxitin were determined by the agar dilution method, according to the CLSI guidelines. Briefly, for each strain, colonies isolated from an overnight growth were transferred to sterile saline; the suspensions were adjusted to a 0.5 McFarland standard and inoculated on Mueller_Hinton agar plates containing 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 or 256mg of cefoxitin per mL using a Steers replicator.

Plates containing Mueller-Hinton agar without antimicrobial were used as controls for bacterial growth.

Polymerase Chain Reaction for mecA Gene

A polymerase chain reaction (PCR) procedure was used to verify the presence of the mecA gene. Primers (mecA₁: 5'-TGG CTA TCG TGT CAC AAT CG and mecA₂: 5'-CTG GAA CTT GTT GAG CAG AG) and protocol used were based on the methodology previously mentioned [8].

ROC Curve

The receiver operator characteristic (ROC) curve was used to determine the best cefoxitin breakpoint to predict oxacillin resistance.

Control Strains

The quality control of the identification tests was done using Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), and Staphylococcus hominis (ATCC 27844). The oxacillin-susceptible S. aureus (ATCC 29213) and oxacillin-resistant S. aureus (ATCC 33591) isolates were used as controls for antimicrobial susceptibility testing and PCR procedure.

Results

A total of 176 CoNS isolates were analyzed. The most frequent species were Staphylococcus epidermidis (120 isolates; 68.2%), followed by Staphylococcus haemolyticus (23 isolates; 13.1%), Staphylococcus capitis (12 isolates; 6.8%), Staphylococcus hominis (6 isolates; 3.4%), Staphylococcus lugdunensis (5 isolates; 2.8%) and other CoNS species (10 isolates; 5.7%).

One hundred and thirty eight (78.4%) CoNS isolates were considered oxacillin-resistant based on the presence of the mecA gene detected by PCR. The presence of the mecA gene, using disk-diffusion method, was predicted with 100% and 98.3% accuracy for cefoxitin and oxacillin disks, respectively (Table 1). Table 1 summarizes the data showing the sensitivity, specificity, negative and positive predictive values and accuracy for the phenotypic tests applied. Using 1µg-oxacillin disk, three isolates (S. epidermidis, S. haemolyticus and S. saprophyticus) that did not harbor the mecA gene were oxacillin resistant. According to the agar dilution method, only one isolate (S. epidermidis) that did not harbor the mecA gene was classified as resistant. The others isolates were classified as susceptible to oxacillin when the follow breakpoints were adopted by us: <8µg cefoxitin per mL and <4µg cefoxitin per mL corresponding to resistant (mecA gene present) and susceptible (mecA gene absence) to oxacillin, respectively. Table 2 presents the discrimination among CoNS isolates in comparison to the presence/absence of the mecA gene and its respective cefoxitin MICs.

Figure 1 indicates receiver operating characteristic (ROC) curves for cefoxitin breakpoints when an agar dilution method is used. At this point, our results showed that 8mg cefoxitin per mL is the best breakpoint to discriminate between CoNS isolates that harbored and did not harbor the mecA gene.

Discussion

The accurate characterization of oxacillin resistance is essential, mainly for correct antimicrobial therapy. Phenotypic tests are important for laboratorial diagnosis. Although molecular methods are considered to be a "gold standard" for the diagnosis of oxacillin resistance, these methods are expensive for many of our laboratories and the use of rapid and accurate phenotypic tests has become an alternative [9].

Since 2004, the CLSI has recommended the use of 30µg-cefoxitin disk for detection of oxacillin resistance among staphylococci species. Presented in the document M100-S15 are the breakpoints for cefoxitin susceptibility (susceptible <8µg cefoxitin per mL, resistant >32µg cefoxitin per mL) although they haven't explicitly specified the mecA gene's presence as the determinant factor for methicillin-resistance. Even thought researches have shown that an agar screening test containing 6µg oxacillin per mL is not efficient towards CoNS [10], comparative studies to asses the agar screening test and disk-diffusion have indicated that agar screening is more sensitive in the detection of methicillin-resistant isolates [11,12]. We have demonstrated that the application of the agar screen test using 4µg cefoxitin per mL as breakpoint showed 99.4% accuracy for oxacillin resistance characterization among CoNS [13]. Despite the same accuracy being demonstrated when 4µg oxacillin per mL was used, cefoxitin agar screen is a feasible method in clinical laboratories and, additionally, it is not necessary to add salt to the culture medium, minimizing errors due to methodological variants.

In this study, cefoxitin breakpoints were evaluated by an agar dilution test, also 30µg-cefoxitin and 1ìg-oxacillin disks, for characterization of oxacillin resistance among CoNS species due to the presence of the mecA gene.

Several studies have showed that the greatest accuracy for oxacillin resistance characterization may be achieved by use of cefoxitin, basically, by the disk-diffusion method [14,15,16]. In our study, 98 and 100% accuracies were obtained with the use of 1µg-oxacillin and 30µg-cefoxtin disks, respectively. In this respect, our results showed that the 30µg-cefoxitin disk presented equal sensitivity but higher specificity than the 1µg-oxacillin disk, confirming the better performance of cefoxitin as a marker of this resistance (mecA-mediated resistance).

Fernandes et al. [17] suggest that a breakpoint of <4mg cefoxitin per mL is accurate for the determination of oxacillin susceptible S. aureus (not harboring mecA gene). The 4µg cefoxitin per mL breakpoint, in our study, showed less accuracy than an 8mg cefoxitin per mL breakpoint (97.1% vs. 99.4%) (Figure 1).

The study described values of >8mg cefoxitin per mL and <4µg cefoxitin per mL as the best breakpoints for characterization of oxacillin -resistant and -susceptible isolates, respectively. Using 8µg cefoxitin per mL, only one S. epidermidis isolate (with MIC of 16mg cefoxitin per mL) was mischaracterized as resistant (Table 2). This breakpoint was shown to be highly reliable in the discrimination of isolates harboring the mecA gene. On the other hand, discrepancies can be observed between isolates harboring and those not harboring the mecA gene because this event (resistance without the presence of the mecA gene) may be associated with the hyperproduction of β-lactamase in these isolates or mutations in PBP's other than PBP 2'. Swenson et al. showed that a cefoxitin test can be used alone to predict mecA-mediated resistance, when these tests were applied for the characterization of this resistance among S. aureus isolates [18,19]. However, despite the excellent concordance between cefoxitin tests and the mecA-gene, non-mecA-mediated resistance may be underestimated [18].

In conclusion, the 30µg-cefoxitin disk was shown to be more specific than the 1mg-oxacillin disk in the prediction of oxacillin resistance due to the mecA gene among CoNS. Also, our results suggested that 8mg cefoxitin per mL is the more accurate breakpoint (99.4%) when an agar dilution method is applied, despite oxacillin resistance (mecA gene) detection not being actually done by it.

Received on 3 December 2007; revised 17 March 2008.

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Publication Dates

• Publication in this collection
  24 Sept 2008
• Date of issue
  June 2008

History

• Received
  03 Dec 2007
• Accepted
  17 Mar 2008