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Journal of Clinical Microbiology, June 2008, p. 2060-2061, Vol. 46, No. 6
0095-1137/08/$08.00+0     doi:10.1128/JCM.02300-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Catalase-Negative Staphylococcus aureus Strain with Point Mutations in the katA Gene{triangledown}

C. Piau,1 J. Jehan,2 R. Leclercq,1* and C. Daurel1

Service de Microbiologie, Université de Caen Basse-Normandie, CHU Côte de Nacre, 14033 Caen cedex,1 Laboratoire de Biologie, Centre hospitalier public du Cotentin—Hôpital de Valognes, 50700 Valognes, France2

Received 29 November 2007/ Returned for modification 7 December 2007/ Accepted 24 March 2008


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ABSTRACT
 
Methicillin-susceptible catalase-negative Staphylococcus aureus strain UCN61 was isolated from an arterial leg ulcer. The deduced sequence of the structural katA gene for the catalase was 99% identical to those of other S. aureus strains. Two mutations were identified in katA from S. aureus UCN61, including one leading to a substitution of key histidine 58 by a tyrosine.


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TEXT
 
The Staphylococcus aureus species expresses various virulence factors, including a catalase which is also considered a key character for genus identification, except for the anaerobic species Staphylococcus saccharolyticus and S. aureus subsp. anaerobius (11). However, some isolates of catalase-negative S. aureus have been previously reported, a few of which have been involved in outbreaks (3, 5).

So far, the molecular basis for loss of catalase activity has been poorly investigated. Only recently, Grüner et al. found that for a clinical isolate of S. aureus, the loss of enzymatic activity was related to a 5-bp deletion leading to a frameshift in the katA (catalase) gene (5).

We report catalase-negative S. aureus subsp. aureus clinical isolate UCN61 and the identification of a point mutation in the katA gene associated with the loss of catalase activity.

A 78-year-old woman was admitted to the General Internal Medicine Department for general impairment and a chronic arterial leg ulcer responsible for several previous hospitalizations. A sample was taken from the superficial inflammatory and painful wound of the right tibia for routine microbiological analysis.

The sample was inoculated on sheep blood agar, Trypticase soy agar (TSA), and MacConkey agar. Sheep blood agar plates were incubated anaerobically and aerobically. After 24 h at 37°C under aerobic atmospheric conditions, beta-hemolytic creamy white colonies of gram-positive cocci were observed on sheep blood agar and TSA. Gram staining showed the presence of round gram-positive cocci in clusters. The catalase test realized with 3% H2O2 was negative. Since the cultural and morphological characteristics were those of staphylococci, the catalase reaction was performed again after subculturing on TSA and chocolate agar incubated under aerobic and CO2-enriched atmospheric conditions, respectively. The test remained repeatedly negative whether using 3% or 30% H2O2. A slide agglutination test (Pastorex; Bio-Rad, Marnes-la-Coquette, France) was weakly positive and a tube coagulase test was positive, leading to the identification of the isolate as S. aureus.

The Vitek2 (bioMérieux, Marcy l'Etoile, France) gram-positive identification card identified the isolate as S. aureus with 93% probability. Only a lack of mannitol and trehalose fermentation was atypical. The negativity of mannitol fermentation was also observed on Chapman medium. The identification of the isolate as S. aureus subsp. aureus was confirmed by sequencing a 463-bp portion of the sodA gene. The sequence displayed 98% identity with S. aureus Mu50 (access number NC_002758.2), S. aureus NCTC 8325 (access number NC_007795.1), and S. aureus COL (access number NC_002951.2) (8).

Antibiotic susceptibility was determined by disk diffusion on Mueller-Hinton agar as recommended by the Comité de l'Antibiogramme de la Société Française de Microbiologie guidelines (2). The strain was susceptible to all routinely tested antibiotics except penicillin G.

To identify the mechanism responsible for lack of catalase activity, the entire katA gene of S. aureus UCN61 was amplified by PCR using two sets of primers (KatDIR1 5'GCGAGTATAGCGCCTCC, KatREV1 5'CAGCAGCTTCTTCATCAG, and KatDIR2 5'CGTCATATGCATGGGTTCG, KatREV2 5'GCCACATTCTGTGCATGC) and sequenced. The primers were designed based on the sequence of the S. aureus Mu50 katA gene. Analysis of the DNA and deduced sequences revealed 99% identity with the corresponding sequences of S. aureus Mu50, NCTC 8325, and COL. Two mutations were identified in S. aureus UCN61: a T172C substitution leading to a histidine 58-to-tyrosine change and a G636A substitution leading to an arginine 212-to-histidine substitution.

Bacteria are exposed to reactive oxygen species from the environment and from those generated by aerobic metabolism. Catalases are heme proteins that detoxify H2O2 by degrading the molecule to water and oxygen. The catalytic reaction occurs in two steps. In the first one, the Fe(III) heme reacts with H2O2 to form an oxoferryl intermediate (compound I). In the second step, compound I reacts with another molecule of H2O2 and the enzyme returns to the resting state (10). The structures of several catalases have been solved, including those of Escherichia coli and Proteus mirabilis, and the resolutions have revealed the presence in the C terminus of the protein of a common highly conserved core. Gouet et al. have determined the tridimensional structure of P. mirabilis catalase and demonstrated that histidine 54 (corresponding to His58 in S. aureus) was essential for the binding of the peroxide group of substrate (4). This histidine is part of a distal pocket composed of three amino acids, tryptophane, arginine, and histidine, that form a major channel (1, 9). Considering its role, His54 was as expected fully conserved among catalases. In E. coli, this key amino acid was located at position 128 in the same conserved environment. His128Ala, His128Asn, and His128Glu mutants have been constructed that showed markedly reduced catalase activity (6). These observations confirm the role of this histidine in catalase activity and lead us to hypothesize that a single histidine mutation was responsible for the lack of catalase activity in strain UCN61.

This report raises two types of problems. First, catalase- and mannitol-negative gram-positive cocci may not be primarily identified as staphylococci in routine analysis. Facing an atypical strain, laboratories should be aware of the risk of misidentification. Second, the virulence of such isolates may be questioned. Many studies suggest that catalase might play a role in S. aureus virulence (7). As a consequence, the lack of catalase activity may result in reduced virulence. In our case, the strain was responsible for a superficial wound infection and its virulence status is therefore difficult to assess.


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ACKNOWLEDGMENTS
 
We thank Peter C. Loewen for helpful discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: CHU de Caen, Service de Microbiologie, avenue Côte de Nacre, 14033 Caen cedex, France. Phone: (33) 02 31 06 45 72. Fax: (33) 02 31 06 45 73. E-mail: leclercq-r{at}chu-caen.fr Back

{triangledown} Published ahead of print on 2 April 2008. Back


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Journal of Clinical Microbiology, June 2008, p. 2060-2061, Vol. 46, No. 6
0095-1137/08/$08.00+0     doi:10.1128/JCM.02300-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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