Eur J Clin Microbiol Infect Dis (2005) 24: 640–642DOI 10.1007/s10096-005-0009-7

BRIEF REPORT

F. Daxboeck . S. Mustafa . O. Assadian . H. Heinzl .M. Stadler . A. M. Hirschl . W. Koller

Accuracy of antibiotyping using standard antibiograms comparedwith 16S–23S ribosomal spacer PCR for diagnosis of MRSA

Published online: 20 September 2005# Springer-Verlag 2005

Molecular methods for typing methicillin-resistant Staphy-lococcus aureus (MRSA) include PCR-based methods,pulsed-field gel electrophoresis (PFGE), spa typing, andmultilocus sequence typing (MLST) [1, 2]. These tech-niques assist in targeted infection control efforts and are,moreover, indispensable for tracking genotypes on a largescale. Nevertheless, in vitro antimicrobial susceptibility pat-terns are still used in epidemiological investigations sinceantibiograms are rapidly delivered to infection controlspecialists without the need for additional investigations.The obvious disadvantage of antibiotyping, however, is thevariability of resistance expression, which is also influ-enced by horizontal transmission and loss of extrachro-mosomal genetic elements. In addition, selective pressure

tends to homogenize the antibiogram pattern over time.Some studies have revealed that by considering thediameters of growth inhibition, it is possible to moreaccurately type MRSA beyond the categorical classifica-tions of susceptible and resistant [3]. However, the doc-umentation and reporting of inhibition zone diametersis not a standard procedure in most hospitals. The aim ofthe study presented here was to assess the positive andnegative predictive values and the sensitivity and specific-ity of antibiotyping MRSA as compared with 16S–23Sribosomal spacer PCR.

The University Hospital Vienna is a 2,140-bed tertiary-care teaching hospital with an MRSA rate of 12.8% [4].From January 2000 to December 2001, 268 patient-uniqueMRSA isolates were collected. The isolates were derivedfrom the following sources: swabs of wounds and screen-ing samples (n=132), respiratory tract (n=50), urine (n=42),intravascular catheter tips (n=17), blood (n=12), andmiscellaneous other clinical materials (n=15).

Staphylococcus aureus was cultured and identifiedaccording to standard procedures. Susceptibility testingfor oxacillin was performed with oxacillin disks (1 μg)according to the guidelines of the Clinical and LaboratoryStandards Institute (CLSI: formerly NCCLS) [5]. Strainsidentified as MRSA were confirmed by multiplex PCRamplification of the S. aureus-specific gene-fragmentSA442 and fragments of the mecA and femB genes [6,7]. Antimicrobial susceptibility testing with erythromycin,clindamycin, gentamicin, amikacin, ciprofloxacin, rifam-picin, and trimethoprim was performed using the Kirby–Bauer disk diffusion method on Mueller–Hinton agaraccording to CLSI guidelines [5]. The compounds fosfo-mycin (120 μg disk) and fusidic acid (10 μg disk), whichcurrently lack CLSI-defined breakpoints for susceptibility,were also included in the study, and the inhibition zonesrequired for susceptibility were ≥10 mm for fosfomycinand ≥14 mm for fusidic acid. For further analysis,intermediate-susceptible and -resistant isolates were uni-formly referred to as non-susceptible isolates. Glycopep-tide antibiotics were not included in the analysis, becauseall of the isolates were susceptible to these compounds.

F. Daxboeck (*)Clinical Institute for Hygiene and Medical Microbiology,Medical University Vienna,Kinderspitalgasse 15,1090 Vienna, Austriae-mail: florian.daxboeck@meduniwien.ac.atTel.: +43-1-404001902Fax: +43-1-404001907

S. MustafaDepartment of Laboratory Medicine,Molecular Biology Division, Medical University Vienna,Währinger Gürtel 18–20,1090 Vienna, Austria

O. Assadian . M. Stadler . W. KollerClinical Institute for Hygiene and Medical Microbiology,Division of Hospital Hygiene, Medical University Vienna,Währinger Gürtel 18–20,1090 Vienna, Austria

H. HeinzlDepartment of Medical Computer Sciences,Medical University Vienna,Währinger Gürtel 18–20,1090 Vienna, Austria

A. M. HirschlClinical Institute for Hygiene and Medical Microbiology,Division of Clinical Microbiology, Medical University Vienna,Währinger Gürtel 18–20,1090 Vienna, Austria

For DNA extraction, two or three colonies were selectedfrom pure culture on Columbia agar and inoculated into5 ml of brain heart infusion broth. After overnightincubation at 35°C, 200 μl of the inoculated brain heartinfusion broth was centrifuged at 1,200 rpm for 5 min andthe supernatant was discarded. To each pellet, 85 μl NETbuffer (10 mM Tris–HCl, pH 7.55; 1 mM EDTA, pH 7.4;10 mM NaCl) and 15 μl achromopeptidase solution(10 U/μl; Sigma-Aldrich, St. Louis, MO, USA) wereadded, and the suspension was shaken for 15 min at 56°C.

For the 16S–23S ribosomal spacer PCR, the master mixcontained a Ready-to-Go (RTG) RAPD Analysis Bead(Amersham Biosciences, Little Chalfont, UK), 25 pmol ofeach primer (G1: 5`-GAA GTC GTA ACA AGG-3′; N1:5′-CAA GGC ATC CAC CGT-3′), 1.4 μl MgCl2 (25 mM),2 μl 5X Rapid-Load (Origene Technologies, Rockville,MD, USA), 2 μl of DNA solution, and 21.1 μl steriledistilled water for a final volume of 25 μl [8, 9]. PCRincluded an initial denaturation step at 95°C for 1 min, 24cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min,and a final extension at 72°C for 1 min. The products of the16S–23S ribosomal spacer PCR were analyzed by electro-phoresis on Polynat 9% Wide Mini S-100 polyacrylamidegels (Elchrom Scientific, Cham, Switzerland) and byethidium-bromide staining.

Simpson’s indices of diversity (SID) for the antibio-grams and the genotypes (as determined by 16S–23Sribosomal spacer PCR) were calculated as describedpreviously [10]. For each genotype comprising more thanfive isolates, a standard antibiogram (SAB) was defined. ASAB was computed as the mode of the genotype-specificantibiogram distribution, i.e., the most frequently observedantibiogram per genotype. Sensitivity, specificity, positiveand negative predictive values (PPV and NPV) of SAB forgenotype (GT) were calculated as follows: sensitivity=n(GT with SAB)/n (GT); specificity=n (no GT with noSAB)/n (no GT); PPV=n (GTwith SAB)/n (SAB); NPV=n(no GT with no SAB)/n (no SAB). Confidence intervals(95%) for proportions were calculated according to themethod of Clopper–Pearson [11].

Among the 268 MRSA isolates studied, 41 differentantibiotypes were observed (SID=0.67). 16S–23S ribo-somal spacer PCR yielded 21 different genotypes

(SID=0.69). Five genotypes comprised >5 isolates desig-nated as A (n=122), C (n=85), E (n=9), G (n=14), and K(n=7). For each of these genotypes, the PPVs of the SABsfor assigning an isolate to the correct genotype (medianPPV, 0.67; range, 0.32–1) and the NPVs for excluding anisolate from a cluster (median NPV, 0.969; range, 0.69–0.985) are shown in Table 1. Sensitivity and specificity ofthe respective SABs for assigning an isolate to the correctgenotype (A, C, E, G, and K) are also shown in Table 1(median sensitivity, 0.43; range, 0.22–0.82; median spec-ificity, 0.976; range, 0.4–1).

Guidelines for the evaluation of microbial epidemiologictyping systems, as published by Struelens [12], recommendcalculating SID to assess the value of a typing method. Inthe present study, the SID of 16S–23S ribosomal spacerPCR was slightly higher than the SID of antibiotyping. It isexpected that the SID of antibiotyping would increase ifmore antibiotics were included in the analysis. However,the application of SID calculation to antibiotyping isproblematic. An identical strain must not yield differenttyping results when well-standardized molecular typingmethods are applied. In contrast, when antibiotyping isperformed, this may be the case due to the development ofsecondary resistance. Therefore, a high SID of antibiotyp-ing is not necessarily indicative of the high value of amethod for epidemiological investigations.

In the present study it was necessary to establish acommon diagnostic study scheme by calculating the PPV/NPV and sensitivity/specificity of antibiotyping. For thispurpose it was necessary to distinguish between diagnostictest-positive and -negative results, which was accom-plished by defining a SAB for each genotype. It is im-portant to point out that this antibiotic susceptibility profilewas expressed by most, but not necessarily the majority, ofisolates belonging to a genotype. The disadvantage of thisSAB classification scheme is that the antibiogram distri-bution may be rather uniform for some genotypes resultingin no prominent mode (e.g. genotype E). In addition, two ofthe genotypes (A, C) in our study had the same SAB.

The present data confirm that molecular typing ofsuspected outbreak isolates with identical antibiograms isadvisable. However, if no molecular methods are available,antibiotyping may be applied in order to exclude potential

Table 1 Positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of respective standard antibiograms(SAB) for assigning an isolate to the correct genotype (GT)

Genotype No. ofGT

SABa No. ofSAB

No. of GTwith SAB

PPV(95%CI)

NPV(95%CI)

Sensitivity(95%CI)

Specificity(95%CI)

A 122 122211211 150 100 0.67 (0.59; 0.74) 0.81 (0.73; 0.88) 0.82 (0.74; 0.88) 0.66 (0.57; 0.73)C 85 122211211 150 48 0.32 (0.25; 0.40) 0.69 (0.59; 0.77) 0.56 (0.45; 0.67) 0.44 (0.37; 0.52)E 9 222221222 2 2 1.0 (0.16; 1.0) 0.974 (0.947; 0.989) 0.22 (0.028; 0.60) 1.0 (0.9859; 1.0)G 14 222211211 12 6 0.50 (0.21; 0.79) 0.969 (0.939; 0.986) 0.43 (0.18; 0.71) 0.976 (0.949; 0.991)K 7 221211212 3 3 1.0 (0.29; 1.0) 0.985 (0.962; 0.996) 0.43 (0.099; 0.82) 1.0 (0.9860; 1.0)Median – – – – 0.67 0.969 0.43 0.976aFor the standard antibiogram, 1=susceptible and 2=non-susceptible for the following antimicrobial agents: amikacin, ciprofloxacin,clindamycin, erythromycin, fosfomycin, fusidic acid, gentamicin, rifampicin, trimethoprim

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outbreak isolates of MRSA according to their in vitrosusceptibility patterns. When using antibiotyping in out-break investigations, though, isolates with identical anti-biograms should not be assigned to the same clusterwithout confirmation by molecular typing.

Acknowledgements F.D. and S.M. contributed equally to thisstudy.

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