1 Microbiology Department, Hospital Gómez-Ulla, Gta. del Ejército s/n, 28007 Madrid, Spain; 2 Microbiology Department, School of Medicine, Universidad Complutense, Avda. Complutense s/n, 28040 Madrid, Spain
Received 4 February 2005; returned 24 May 2005; revised 30 May 2005; accepted 2 June 2005
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Abstract |
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Methods: Seventeen clinical isolates of S. lugdunensis (identified by PCR amplification and sequencing of the rpoB gene) were studied using the ATB32-Staph, Crystal, Vitek 2 and Wider commercial systems. The clumping factor test and the tube coagulase test were also performed. ß-Lactamase production was studied by chromogenic methods. Methicillin resistance was phenotypically studied by the MRSA slide latex agglutination test, growth in MRSA agar, and the Vitek 2 and Wider systems (based on oxacillin MIC), and genotypically studied by detection of the mecA gene by PCR.
Results: The clumping factor test was negative in 35.3% of strains. All isolates were correctly identified to species level by the ATB32-Staph system. Species misidentification rates were 5.9%, 23.5% and 29.4% with the Crystal, the Vitek 2 and the Wider systems, respectively, mostly as Staphylococcus haemolyticus. ß-Lactamase was present in 11.8% of strains. Whereas 76.5% and 47.1% of strains exhibited oxacillin resistance (MIC range 0.52 mg/L) by the Vitek 2 system and the Wider system, respectively, none of the strains was positive in the MRSA slide latex agglutination test or grew in MRSA agar. All strains lacked the mecA gene.
Conclusions: The clumping factor test and some commercial systems may misidentify S. lugdunensis. Oxacillin resistance detected by commercial systems is not indicative of the presence of the mecA gene. These facts, together with ß-lactamase production, may preclude adequate treatment of infections by this virulent coagulase-negative Staphylococcus.
Keywords: S. lugdunensis , methicillin resistance , ß-lactamases , mecA gene , clumping factor
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Introduction |
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With respect to identification, the reference method of Kloos and Schleifer10 is considered too complicated and too lengthy to be used in routine practice.11 On the other hand, commercial systems, designed to identify all coagulase-negative species (clinical, veterinary and alimentary), are not very specific, lack sufficient information in their database or show variable results when compared with other systems.11 Most reports of S. lugdunensis infections comment on problems of false-positive test results for clumping factor that are suggestive of S. aureus,12 although S. lugdunensis typically gives negative results in the tube test for free coagulase. Ornithine decarboxylase (ODC), pyrrolidonyl arylamidase (PYR) and the generation of acid from D-mannose are crucial in the identification of S. lugdunensis.11,13
With respect to susceptibility, S. lugdunensis usually lacks both the mecA gene (encoding PBP 2a and methicillin resistance) and ß-lactamase, and is thus susceptible to penicillin.14 In addition, some reports indicate that most strains are susceptible to all antibiotics,1 a characteristic that, together with ODC, has been used as a screening method for S. lugdunensis.7 However, false-positive oxacillin MIC results (versus negative detection of the mecA gene by PCR) have been described in commercial systems.15 This has led to the suggestion that S. lugdunensis should be excluded from the Vitek system to enhance its specificity,16 and to the fact that the NCCLS emphasizes that current interpretative criteria for oxacillin MICs could be misleading in strains of this virulent pathogenic Gram-positive coccus.17
These facts stress the need for accuracy in identification of S. lugdunensis clinical isolates, and their associated antibiotic susceptibility profile. Thus, this study was carried out to compare different methods of susceptibility determination after adequate identification of S. lugdunensis isolates.
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Materials and methods |
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Eighteen Gram-positive cocci, suspected to belong to the lugdunensis species of the genus Staphylococcus (colony morphology, catalase positive, tube coagulase negative and ODC negative), isolated from 18 clinical samples (11 from abscesses, three from infected wounds, two from blood cultures, one from a catheter and one from urine) received in the Microbiology Department during January 1997December 2004, were studied.
Reference strains
S. lugdunensis ATCC 6343, S. aureus ATCC 29213 (methicillin-susceptible) and a methicillin-resistant S. aureus (MRSA) clinical isolate were used as control strains.
Identification methods
Genotypic characterization. Amplification of the rpoB gene was performed by PCR as previously described,18 and subsequent sequencing reactions were carried out with the reagents of the ABI Prism dRhodamine dye terminator cycle sequencing ready reaction kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA). Sequences obtained were compared through the National Centre for Biotechnology Information (NCBI) BLAST Network Service.
Phenotypic characterization
Four commercial identification systems were used in the study: (i) ATB32-Staph (bioMérieux, Marcy l'Etoile, France); (ii) Crystal manual system with a Gram-positive (GP) card (Becton Dickinson, Sparks, MD, USA); (iii) automatic Vitek 2 system with a Gram-positive susceptibility card (bioMérieux); and (iv) semi-automatic Wider system (Soria Melguizo S.A., Madrid, Spain) with a W3 card (Dade Behring, West Sacramento, CA, USA). Results from tests were considered atypical when discrepancies were found in comparison with the commercial system database. In addition, the clumping factor test (Pastorex Staph-Plus, Bio-Rad, Marnes-la-Coquette, France) for detection of bound coagulase was performed.
Susceptibility tests
Genotypic characterization. Disc diffusion of cefoxitin (0.03 mg) as a mecA induction method19 was used. mecA gene determination by PCR with specific primers was performed as previously described.20
Phenotypic characterization
Four methods were used for phenotypic detection of methicillin resistance: (i) the MRSA slide latex agglutination test (Slide MRSA Detection test; bioMérieux); (ii) growth on MuellerHinton agar (Becton Dickinson) supplemented with 4% NaCl, with 6 mg/L oxacillin (MRSA agar) and 24 h of incubation at 35°C; (iii) Vitek 2 system with a GPI-523 card (bioMérieux); and (iv) Wider system with a W3 card.
In addition, ß-lactamase determination was performed with a chromogenic cephalosporin substrate (Cefinase, Becton Dickinson).
Statistical analysis
The Fisher's Exact test was used to compare the detection of methicillin resistance by the different methods. Oxacillin MIC 0.5 mg/L21 was considered an indicator of methicillin resistance in the Vitek 2 and Wider systems. A P < 0.01 was considered significant.
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Results |
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S. aureus ATCC 29213 and the MRSA reference strain were clumping factor and tube coagulase test positive, and the specific sequence of the rpoB gene for S. lugdunensis was not detected by PCR.
All 17 S. lugdunensis strains were free coagulase negative. Bound coagulase (clumping factor) was positive in all but six strains (strains 9, 10, 13, 14, 15 and 16). Clumping-factor negative strains represented 35.3% of strains.
Table 1 shows the phenotypic biochemical identification of the 17 S. lugdunensis strains plus the ATCC 6343 strain. ATB32-Staph showed an absolute concordance between S. lugdunensis identification and the presence of the specific sequence of the rpoB gene. The Crystal system misidentified strain 3 as S. haemolyticus, but all other strains except two presented atypical tests, mainly L-arabinose and maltotriose. A total of 23.5% of isolates (four out of 17) were misidentified as Staphylococcus haemolyticus by the Vitek 2 system, and 29.4% of isolates (five out of 17) were misidentified as S. haemolyticus (two strains) and Staphylococcus auricularis (three strains) by the Wider system.
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Table 2 shows the results of susceptibility testing of the isolates. No differences were found between absence of the mecA gene (100% of strains), negative MRSA slide latex agglutination tests (100% of strains) and absence of growth in MRSA agar (100% of strains). When identifying methicillin resistance by oxacillin MIC 0.5 mg/L, 76.5% (13 out of 17) and 47.1% (eight out of 17) of strains were considered resistant by the Vitek 2 system and the Wider system (P = 0.15), respectively. Logically, significant differences (P < 0.001) were found between the resistance detected by these systems (>45%) and the resistance detected by the MRSA slide latex agglutination test, growth in MRSA agar or presence of the mecA gene (0%). More strains with higher MIC values (oxacillin MIC = 2 mg/L) were found by the Wider system (eight strains) versus the Vitek 2 system (three strains) (P = 0.14).
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Discussion |
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It has been suggested that S. lugdunensis should be suspected when a staphylococcal isolate gives a positive reaction for clumping factor (bound coagulase) but a negative reaction in the tube coagulase test (free coagulase).22 All isolates in reports from the USA (including 20 isolates)23 and from Spain (including 25 isolates)24 were clumping-factor positive. The fact that 35.3% (six out of 17) of our S. lugdunensis isolates were clumping-factor negative excludes the exclusive use of this test as a base for suspicion. While the presence of clumping factor can easily lead to misidentification as S. aureus,24 from which S. lugdunensis can be differentiated by the tube coagulase test, the absence of clumping factor in approximately one-third of our S. lugdunensis isolates may exclude the degree of suspicion needed, resulting in a failure to microbiologically diagnose this virulent pathogen in an important number of cases. Standard commercial systems do not solve the problem in some of the cases since 29.4%, 23.5% and 5.9% of our isolates were misidentified by the Wider, Vitek 2 and Crystal systems, respectively. All isolates were identified as S. lugdunensis by the ATB32-Staph system, with 100% concordance with PCR detection of the rpoB specific sequence. PCR is very sensitive and can be considered the gold standard, but is not commonly used in many routine laboratories. Thus, ATB32-Staph seems adequate for the identification of S. lugdunensis on a routine basis. A rapid and inexpensive working five-test simple scheme (novobiocin, urease, D-mannose, PYR and ODC) has been found to be useful in a series of 12 S. lugdunensis11 and may be more cost-effective than ATB32-Staph.
It has been suggested that penicillin resistance is rare in S. lugdunensis due to the lack of ß-lactamase and the mecA gene. With respect to ß-lactamase, around 4% of strains in Spain presented a positive chromogenic test.5 In our series, 11.8% (two out of 17) of strains were penicillin resistant due to ß-lactamase production.
The gold standard for detection of methicillin resistance is the detection of the mecA gene by PCR, again unsuitable for routine testing.15 In all 17 clinical isolates (mecA negative), there was a 100% correlation with the negative MRSA slide latex agglutination test or with absence of growth in MRSA agar. Other authors have shown very good accuracy of the MRSA slide latex agglutination test for the detection of PBP 2a in coagulase-negative staphylococci, with very high specificity and sensitivity.25 Problems arise when determining oxacillin MICs for the detection of methicillin resistance with commercial systems such as Vitek 2 or Wider, since 76.5% and 47.1% of the 17 mecA negative S. lugdunensis strains in this series exhibited oxacillin resistance (MIC 0.5 mg/L),21 respectively. It has been considered that S. lugdunensis isolates with oxacillin MICs
0.5 mg/L should be examined for the presence of PBP 2a or the mecA gene.23 In our series, all strains with MIC values 0.52 mg/L (13 and eight strains out of 17 with the Vitek 2 and Wider systems, respectively) were mecA negative. This is in agreement with other authors, who have suggested that isolates exhibiting oxacillin MICs of 0.52 mg/L usually lack the mecA gene and thus should be considered susceptible.14 When methicillin resistance is present, as in a recently described clinical isolate of S. lugdunensis carrying the mecA gene, the oxacillin MIC was >256 mg/L.22 This strain would surely grow in MRSA agar and would be positive in the MRSA slide latex agglutination test. From the results obtained in our series, negative results in these two tests relate to absence of mecA.
ß-Lactamase production in some strains of S. lugdunensis in our country, and the report of penicillin resistance due to the presence of mecA gene in one isolate in Singapore,22 preclude the generalization of universal penicillin and cephalosporin susceptibility in S. lugdunensis. This is important because commercial methods for detection of oxacillin susceptibility gave confusing results (artificially increasing methicillin resistance prevalence since isolates with oxacillin MICs 0.52 mg/L in this series lacked the mecA gene). Thus there is a need for accurate and rapid identification of S. lugdunensis, not basing suspicion only on rapid tests such as clumping factor because of the possibility (35.3% in this series) of strains negative for this factor.
There is a lack of clinical information on penicillin treatment of infections caused by S. lugdunensis that have high oxacillin MICs (i.e. 2 mg/L) but lack the mecA gene. Further studies, such as pharmacodynamic simulations/animal models are needed to explore if these in vitro facts have in vivo consequences, with respect to penicillin treatment.
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Acknowledgements |
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