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Journal of Clinical Microbiology, November 2008, p. 3829-3832, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.01440-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Thymidine-Dependent Staphylococcus aureus Small-Colony Variants: Human Pathogens That Are Relevant Not Only in Cases of Cystic Fibrosis Lung Disease {triangledown}

Silke Besier,1* Johannes Zander,1 Ekkehard Siegel,2 Stephan H. Saum,3 Klaus-Peter Hunfeld,1 Annabelle Ehrhart,1 Volker Brade,1 and Thomas A. Wichelhaus1

Institute of Medical Microbiology and Infection Control, Hospital of Johann Wolfgang Goethe-University, Frankfurt/Main, Germany,1 Institute of Medical Microbiology, Hospital of Johannes Gutenberg-University, Mainz, Germany,2 Institute of Molecular Biosciences, University, Frankfurt/Main, Germany3

Received 28 July 2008/ Returned for modification 31 August 2008/ Accepted 20 September 2008


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ABSTRACT
 
We report the isolation of thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus from unusual infection sites of patients with chronic soft tissue infection, tympanitis, bronchitis, peritonitis, and septicemia. Furthermore, we provide evidence that the essential growth factor for TD-SCVs, i.e., thymidine, and its metabolite dTMP are present in various human specimens.


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TEXT
 
During chronic Staphylococcus aureus infections, variants of this pathogen can emerge that are able to persist in patients for months or years in a dormant metabolic state (14). These bacteria are designated "small-colony variants" (SCVs) due to their phenotypic traits. In contrast to "normal-colony variants" (NCVs), SCVs grow on sheep blood agar as tiny, nonpigmented, and nonhemolytic colonies and show decreased metabolic activity (10, 11, 12, 19). SCVs are easily overgrown by more rapidly dividing strains (14) and can be cultured only in the presence of distinct growth factors. While SCVs dependent on hemin or menadione can be isolated from patients with osteomyelitis, persistent soft tissue infection, and device-related infections (15, 17, 20, 21), thymidine-dependent SCVs (TD-SCVs) are well-known in the context of cystic fibrosis (CF) lung disease, especially after prior use of trimethoprim-sulfamethoxazole (SXT) (2, 7, 9). It has been shown recently that random mutations in the thymidylate synthase-encoding thyA gene which lead to an intracellular lack of dTMP are responsible for the formation of the TD-SCV phenotype (1, 4, 24) and that hypermutability due to a defect DNA mismatch repair system favors the acquisition of these mutations (3). As TD-SCVs are apparently able to use thymidine and/or the metabolite dTMP from the environment, these variants are able to bypass the antibiotic effect of folic acid antagonists and consequently are resistant to SXT (7, 13, 24).

Widely unknown, however, is the occurrence of TD-SCVs in clinical specimens other than CF respiratory samples. In the following, we describe five cases with isolation of TD-SCVs from unusual infection sites and the characteristics of the corresponding isolates, and we analyze for the first time the thymidine and dTMP concentrations in various human specimens.

Characteristics of patients with isolation of TD-SCVs. The TD-SCVs were recovered from the microbiological specimens of five patients (A to E) (Table 1) attending the University Hospitals of Mainz and Frankfurt/Main, Germany, between January 2005 and February 2008. The patients were of different ages and both genders and all of them had a chronic underlying disease, a genetic disorder, i.e., CF (patients B and D) and Nijmegen breakage syndrome (patient A), a hematooncological disease (patient C), or chronic otitis media (patient E). In addition, all patients suffered from chronic infections, comprising soft tissue infection (patient A), recurrent abscess with subsequent peritonitis (patient B), chronic bronchitis (patient C), relapsing septicemia (patient D), and tympanitis (patient E). In spite of interventional antimicrobial therapy, clinical signs of infection persisted in four patients (A, C, D, and E) over several weeks up to several months. Only patient B was cured shortly after surgical intervention. The two CF patients (B and D) harbored additionally isogenic TD-SCV isolates in their respiratory samples, as verified by pulsed-field gel electrophoresis (PFGE) (22) (Fig. 1). With regard to patient B, a strain with clonal identity to the intraabdominal wound swab isolate was found 4 months after the peritonitis, whereas in the context of patient D, a strain isogenic to the blood culture isolate was already detected 1 year before the septicemia. All five patients had received prior oral long-term prophylaxis with SXT.


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  		TABLE 1. Characteristics of patientsa


Figure 1
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  		FIG. 1. PFGE patterns of SmaI digests of total DNA from clinical TD-SCV isolates (S105, S284, S349, S286, S291, and S200) and corresponding revertants with NCV phenotype (S327, S328, S329, S330, S331, S332, S333, S334, S335, S336, S337, and S338). The intraabdominal wound swab isolate S284 and respiratory isolate S349 were isolated from CF patient B, whereas blood culture isolate S291 and the respiratory isolate S200 were derived from CF patient D. Lane M, DNA size markers. Molecular sizes of the marker fragments are given in kilobases on the left.

Characteristics of TD-SCV isolates with regard to NCV reversion, alterations in thyA, and antibiotic susceptibility. To generate revertants with an NCV phenotype, 10 ml of brain heart infusion broth was inoculated with one colony of the appropriate TD-SCV isolate, grown overnight with shaking (200 rpm) at 37°C, and streaked onto Mueller-Hinton agar. Variants growing on Mueller-Hinton agar after overnight incubation at 37°C were subcultivated on blood agar and designated NCV revertants if they displayed the appropriate characteristics (2). The experiment was performed in triplicate and showed that four of the five TD-SCV isolates were able to revert to the NCV phenotype during subcultivation, depending on the extent of the alterations in thyA (Table 2). In this context, sequence analysis revealed that, in line with TD-SCVs isolated from CF respiratory samples, TD-SCVs isolated from unusual infection sites harbored various mutations in thyA, comprising individual nucleotide substitutions that resulted in premature stop codons and amino acid substitutions as well as a 9-bp, in-frame deletion (1, 4). Furthermore, it became evident that an SCV-inducing nonsense mutation can be compensated for by different missense mutations in the same codon. Only TD-SCV S286 with an SCV-inducing missense mutation showed reversion to the wild-type sequence in all three NCV revertants. Clonal identity of TD-SCVs and NCV revertants was proven by PFGE (Fig. 1). MIC determination by the use of Etest strips showed that TD-SCVs and corresponding revertants had similar MICs for amoxicillin, oxacillin, gentamicin, clarithromycin, levofloxacin, tetracycline, fosfomycin, and rifampin, irrespective of the mutations in thyA. All isolates were oxacillin susceptible and merely SXT was interpreted as being resistant for all TD-SCVs (MICs > 32 µg/ml) and susceptible for all revertants (MIC range, 0.064 to 0.125 µg/ml) according to CLSI guidelines (5).


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  		TABLE 2. Mutations affecting the thyA gene of clinical S. aureus TD-SCVs and variety of compensatory thyA mutations in spontaneous revertants with normal colony appearance

Concentrations of thymidine and dTMP in human specimens. Although it has been postulated that thymidine is abundant in clinical specimens with necrotic cells (13, 14), only human blood and plasma have been analyzed so far, and the thymidine concentrations found were relatively low (6, 8, 18). To determine the thymidine and dTMP concentrations in various human specimens, i.e., CF sputum, pus, urine, liquor, and blood, we added 100 µl of the appropriate sample to 900 µl of 77% methanol, sonicated for 15 min at 4°C, and centrifuged for 15 min at 13,000 x g. The supernatant was sterile filtered by use of a 0.22-µm-pore-size filter and separated by high-performance liquid chromatography using a 5-µm ACE 5 C18 column with a length of 250 mm and an internal diameter of 4.6 mm (LCC Engineering and Trading GmbH, Egerkingen, Switzerland). Solvents and elution conditions were applied in accordance with Yu et al. (23). For each kind of specimen, samples from three different patients were analyzed (Table 3). Thymidine was found in CF sputum and urine, but the nucleoside was not detectable in pus, liquor, and blood by the high-performance liquid chromatography method within the detection limit. In contrast, dTMP, a metabolite of thymidine, was found in most of the samples analyzed. Very high concentrations of dTMP were determined for specimens with large amounts of damaged tissue, i.e., sputum samples from CF patients and pus aspirate samples. Lower concentrations of dTMP were found in urine and liquor samples, and no dTMP was detectable in human blood. To the best of our knowledge, the observation that a metabolite of thymidine, i.e., dTMP, and not thymidine itself is abundant in human specimens with necrotic cells has not been described so far. This finding is of crucial importance, since proliferating TD-SCVs of S. aureus have been demonstrated to utilize extracellular dTMP as a growth factor in vitro (24).


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  		TABLE 3. Concentration of thymidine and dTMP in various human specimens

Conclusions. A comparison of the data presented here with literature provides direct evidence that the isolation of S. aureus TD-SCVs is not a phenomenon restricted to cases of CF lung disease (2, 7, 9, 16). This special phenotype is able to survive in all clinical specimens that contain thymidine or its metabolite dTMP. A prophylaxis or therapy with SXT apparently favors the emergence of TD-SCVs due to a selection of detrimental mutations in thyA. Thus, all clinical specimens of patients with chronic infections and anamnesis of SXT treatment should be carefully screened for the presence of TD-SCVs.


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ACKNOWLEDGMENTS
 
We thank Denia Frank for excellent technical assistance.

This work was supported in part by a financial grant from Mukoviszidose e.V., Bonn, Germany, the German Cystic Fibrosis Association.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Microbiology and Infection Control, Hospital of Johann Wolfgang Goethe-University, Frankfurt/Main, Paul-Ehrlich-Straße 40, 60596 Frankfurt/Main, Germany. Phone: 49 69 6301 6438. Fax: 49 69 6301 5767. E-mail: s.besier{at}em.uni-frankfurt.de Back

{triangledown} Published ahead of print on 1 October 2008. Back


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Journal of Clinical Microbiology, November 2008, p. 3829-3832, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.01440-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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