Prevalence and genetic diversity of Staphylococcus aureus small-colony variants in cystic fibrosis patients


Corresponding author: S. Yagci, Fakulteler M. Keskin S. 9/3 Cebeci, 06590 Cankaya/Ankara, Turkey


Staphylococcus aureus small-colony variants (SCVs) are being isolated more frequently in cystic fibrosis (CF) patients. We aimed to determine the prevalence of S. aureus SCVs and their phenotypic and genotypic properties in CF patients admitted to a university hospital. Specimens of 248 patients were examined during a period of 11 months. Colonies supposed to be SCVs were evaluated on Columbia blood agar, mannitol salt agar, and brain–heart infusion agar with 5% NaCl (BHIA 5% NaCl). Strains were confirmed by S. aureus nucA PCR. Antibiotic susceptibilities of SCVs and simultaneously isolated S. aureus strains were determined for oxacillin, gentamicin, trimethoprim–sulphamethoxazole, vancomycin, ciprofloxacin, linezolid, and tigecycline. Genetic relatedness between SCVs and normal S. aureus strains was determined with a pulsed-field gel electrophoresis (PFGE) method. S. aureus SCVs were detected in 20 of 248 patients (8.1%). The highest SCV isolation rate was obtained with MSA, followed by BHIA 5% NaCl. Auxotrophism for thymidine was demonstrated in six SCVs. The tigecycline susceptibilities of 48 SCV strains isolated in this study showed higher MIC values than those of 33 simultaneously isolated normal S. aureus strains. Whereas SCVs and normal S. aureus strains showed identical genotypes in 14 of the patients, five patients showed different genotypes. This first study from Turkey evaluating S. aureus SCVs in CF patients has indicated the importance of considering and reporting SCVs in chronic infections such as CF. The presence of SCVs will probably indicate persistent infection, and this might impact on antibiotic treatment decisions, as they are more resistant to antibiotics.


Cystic fibrosis (CF) patients are often colonized with Staphylococcus aureus. The spectrum of microorganisms isolated from the respiratory tract specimens of CF patients has changed in recent years, and, as well as the frequent pathogens S. aureus and Pseudomonas aeruginosa, Burkholderia cepacia complex, Stenotrophomonas maltophilia, Achromobacter xylosoxidans and S. aureus small-colony variants (SCVs) are being isolated more frequently [1–3]. S. aureus SCVs are known as slow-growing subpopulations of S. aureus that grow as small, non-pigmented and non-haemolytic colonies on agar plates, and their isolation and identification is a challenge for clinical microbiology laboratories [4]. This difficulty may lead to diagnostic underestimation and therefore therapeutic failures in the clinical setting. S. aureus SCVs have been associated with chronic and recurrent infections; however, this relationship has only been appreciated in recent years [5,6].

The discovery and characterization of S. aureus SCVs have provided new insights into our understanding of the pathogenesis of chronic S. aureus lung infections in CF patients. As SCVs can survive intracellularly, the isolation of an SCV phenotype is usually associated with the persistence of S. aureus in CF patients. This study was aimed at determining the prevalence and antibiotic susceptibility profiles of S. aureus SCVs in CF patients, identifying the best method for detection of SCVs in clinical specimens, and evaluating the genetic relatedness between normal S. aureus strains isolated from CF patients and their paired SCV strains.

Materials and Methods

This prospective study was conducted at Hacettepe University Children’s Hospital, Chest Diseases Unit and Clinical Microbiology Laboratory, Ankara, Turkey between February 2007 and January 2008. Respiratory specimens of 248 CF patients were screened for the presence of S. aureus SCVs. The specimens comprised sputum and deep throat swab samples.

Specimens were cultured on Columbia sheep blood agar (BD, Franklin Lakes, NJ, USA) and chocolate agar (Fluka, Sigma-Aldrich, St. Louis, MO, USA) with 300 U/mL bacitracin (Sigma-Aldrich, St. Louis, MO, USA), Eosin Methylene Blue agar (Becton Dickinson), mannitol salt agar (MSA) (Becton Dickinson), oxidation/fermentation polymyxin–bacitracin–lactose agar, and brain–heart infusion agar (Oxoid, Basingstoke, UK) with 5% NaCl (Merck, Darmstadt, Germany) (BHIA 5% NaCl). All agar plates were incubated at 35°C for at least 48 h aerobically, except for BHIA 5% NaCl plates, which were incubated anaerobically.

Colonies suspected of being S. aureus on sheep blood agar, MSA and BHIA 5% NaCl were further isolated at 35°C for 24–48 h on Columbia sheep blood agar. Non-haemolytic, non-pigmented, pinpoint or fried-egg colonies on sheep blood agar and small colonies on MSA and BHIA 5% NaCl were considered to be S. aureus SCVs. These suspected colonies were inoculated onto Columbia blood agar and Schaedler agar (Becton Dickinson). Columbia blood agar was incubated in a normal atmosphere and Schaedler agar was incubated in 5–10% CO2, both at 35°C. If the colonies were observed to be normal-sized, haemolytic and pigmented on Schaedler agar, they were considered to be S. aureus SCVs. Strains were subjected to species identification by Gram staining, catalase reaction, tube coagulase result, and latex agglutination test (Slidex Staph Plus; bioMérieux, Marcy l’Etoile, France). Identification of S. aureus was confirmed by nucA PCR [7].

Auxotrophy for haemin (5.4 μg) was tested by using standard disks (Sigma), and auxotrophy for thymidine and menadione was tested by impregnating disks with 1.5 μg of thymidine (Sigma) or 1.5 μg of menadione (Sigma), respectively. A strain was positive for auxotrophy if a zone of growth surrounding the impregnated disks on Mueller–Hinton agar was detected after 24 h of incubation at 35°C [8,9].

Antimicrobial susceptibilities of the strains were determined by the broth microdilution method for oxacillin, gentamicin, vancomycin, ciprofloxacin, linezolid, trimethoprim–sulphamethoxazole and tigecycline according to CLSI guidelines [10]. Strains with a normal phenotype were tested on Mueller–Hinton broth, and SCVs were tested on brain–heart infusion broth [11]. The MIC values of SCVs were evaluated according to CLSI breakpoints for staphylococci [12]. As there are no CLSI criteria for tigecycline, EUCAST susceptibility criteria were applied [13].

Clonal identity and genetic relatedness between normal S. aureus strains and their paired SCVs were analysed with a pulsed-field gel electrophoresis (PFGE) method after SmaI (Sigma) restriction of bacterial DNA, as described previously [14]. The 33 normal S. aureus strains selected for PFGE were the pairs of related SCVs isolated from each patient. The PFGE bands produced were evaluated according to the Tenover criteria [15]. PFGE genotypes were numbered separately within each patient. No interpatient genetic relatedness analysis was performed.

Clinical data of patients with SCV isolation were collected and evaluated from patients’ medical records by the physicians of the paediatric chest diseases unit. Statistical analysis was performed with SPSS software, version 15.0. Definitive statistics were represented as means, medians, and percentages. Differences between means were evaluated with the Mann–Whitney test for the numerical variables and the chi-square, Mantel–Haenzel or Fisher exact test for the categorical variables. A p-value cut-off of ≤0.05 was considered to be statistically significant for all analyses.


A total of 519 respiratory specimens from 248 CF patients were evaluated for the presence of S. aureus SCVs. Of these 519 specimens, 209 (40.3%) were sputum and 310 (59.7%) were deep throat swab samples. The number of male patients (129, 52%) was similar to the number of female patients (119, 48%). The median age of patients was 9.9 years (range: 1–58 years), and was similar in the two genders (Table 1).

Table 1.   Comparison of the ages of patients with normal Staphylococcus aureus and the ages of patients with S. aureus small-colony variants (SCVs)
Age (years)Patients with normal S. aureusPatients with S. aureus SCVs
n (%) n (%)
1.0–5.042 (34.1)3 (15.0)
6.0–10.031 (25.2)4 (20.0)
11.0–15.023 (18.7)5 (25.0)
16.0–20.017 (13.8)3 (15.0)
21.0–25.05 (4.1)4 (20.0)
≥26.05 (4.1)1 (5.0)
Total123 (100.0)20 (100.0)

Of the 248 patients, 123 (49.6%) harboured normal S. aureus strains in their respiratory specimens, and 20 (8.1%) harboured SCVs as well. The prevalence of SCVs in S. aureus-positive patients was 16.2% (20/123). Of these 20 SCV-positive patients, 13 (65%) were females and seven were males. The median age of patients with normal S. aureus strains was 10.4 years (range: 1–58 years), and that of patients with SCVs was 14.4 years (range: 2–31 years); the isolation rate was highest in those between 11 and 15 years of age, who constituted a total of 25% of the patients (p <0.05). Patients with SCVs were older than patients with normal S. aureus strains only (p 0.015, Mann–Whitney U-test) (Table 1). Among the 20 SCV carriers, 13 had SCVs once, two had SCVs twice, and five had SCVs three times. Persistent colonization was defined as three or more positive cultures during the study period, and was demonstrated in 25% (= 5) of SCV carriers. From these 20 patients, 48 SCVs and 30 normal S. aureus strains were isolated in a total of 32 samples. Fifteen (46.9%) of these 32 samples also revealed the simultaneous presence of P. aeruginosa and S. aureus SCVs.

The review of patients’ medical reports revealed that no antistaphylococcal prophylaxis had been given to the patients. The most frequently used antibiotics for the staphylococcal infections in CF patients were amoxycillin–clavulanate, ampicillin–sulbactam, and fluoroquinolones. Trimethoprim–sulphamethoxazole was used in only five patients and gentamicin in four patients. All of the five patients with SCVs with trimethoprim–sulphamethoxazole resistance had been treated with trimethoprim–sulphamethoxazole. The length of the treatment varied from 1 month to 2 years for different patients. The trimethoprim–sulphamethoxazole-resistant SCVs from these five patients were thymidine-dependent SCVs (Table 2).

Table 2.   Genetic diversity of Staphylococcus aureus small-colony variants (SCVs) and paired S. aureus strains from 20 patients harbouring S. aureus SCVs
Patient no.Date of admissionPhenotypePFGE genotypeOXGMVACIPLZSXTTG
  1. PFGE, pulsed-field gel electrophoresis; OX, oxacillin; GM, gentamicin; VA, vancomycin; CIP, ciprofloxacin; LZ, linezolid; SXT, trimethoprim–sulphamethoxazole; TG, tigecycline; R, resistant; I, intermediate; S, sensitive.

  2. PFGE genotypes are numbered separately for each patient. No interpatient genetic relatedness analysis was performed.

 16 March 2007SCVIIRRSRSRR
S. aureus ISSSSSSS
 217 April 2007SCVISSSISSS
3 August 2007SCVISSSSSSS
19 November 2007 S. aureus IVSSSSSSS
 318 April 2007 S. aureus ISSSSSSS
 426 April 2007 S. aureus IRSSISSS
7 June 2007 S. aureus ISSSSSSS
17 December 2007 S. aureus ISSSSSSS
S. aureus IRSSSSSS
 530 March 2007 S. aureus ISSSSSSS
24 October 2007 S. aureus IRSSSSSS
11 December 2007 S. aureus ISSSSSSS
 65 June 2007SCVISSSISSS
S. aureus ISSSISSS
26 September 2007SCVISSSISSS
17 January 2008 S. aureus ISSSISSS
 75 June 2007 S. aureus ISSSRSSS
 85 June 2007 S. aureus ISSSRSSS
 927 June 2007 S. aureus IISSSISSS
17 August 2007 S. aureus IISSSSSRS
8 October 2007 S. aureus IIRSSSSSS
S. aureus ISSSISSS
104 July 2007 S. aureus IIRSSSSSS
S. aureus ISSSSSSS
1127 June 2007 S. aureus ISSSSSSS
123 August 2007 S. aureus ISSSRSSS
1322 August 2007SCVISSSSSSS
S. aureus ISSSISSS
141 October 2007 S. aureus IRSSRSSS
7 January 2008 S. aureus IRSSRSSS
1516 October 2007 S. aureus ISSSSSSS
1615 November 2007 S. aureus IRSSSSSS
1711 December 2007 S. aureus IRSSSSSS
S. aureus ISSSSSSS
1810 December 2007SCVIRSSSSSR
1929 November 2007 S. aureus ISSSRSSS
2031 January 2008 S. aureus ISSSSSSS

Of the 48 SCVs, 28 were isolated after an incubation period of 24 h and 20 after an incubation period of 48 h. The highest SCV yield was from MSA (66.7%), followed by BHIA 5% NaCl (50%). For patients infected or colonized with SCVs, a reliable bacterial count could not be obtained, as they grow as tiny colonies and are usually covered by normal S. aureus colonies. Typical small colonies were observed for 36 (75%) of strains (Fig. 1). Six strains (12.5%) formed pinpoint colonies and six (12.5%) formed fried-egg colonies.

Figure 1.

Staphylococcus aureus small-colony variants (black arrow) and normal S. aureus colonies (red arrow) on mannitol salt agar (left) and brain–heart infusion agar with 5% NaCl (right).

Among the 48 SCVs, six were thymidine-dependent (Fig. 2). Of these six, one was also haemin-dependent. Five of the thymidine-dependent strains formed fried-egg colonies. No menadione-dependent strains were detected.

Figure 2.

 Thymidine-dependent Staphylococcus aureus small-colony variants on Mueller–Hinton agar. H, haemin; M, menadione; T, thymidine.

The resistance rates of SCVs were as follows: oxacillin, 10.4%; trimethoprim–sulphamethoxazole, 16.7%; ciprofloxacin, 14.6%; and gentamicin, 6.3%. When compared with those for simultaneously isolated normal S. aureus strains, these resistance rates showed no significant difference. No resistance was detected for vancomycin and linezolid. MICs for tigecyline were significantly higher for SCVs than for simultaneously isolated normal S. aureus strains (p 0.041, Fisher) (Table 3).

Table 3.   Antibiotic susceptibilities of Staphylococcus aureus small-colony variants (SCVs) and paired normal S. aureus strains
Antibiotic S. aureus (n = 33) S. aureus SCV (n = 48)
MIC range (mg/L)MIC50 (mg/L)MIC90 (mg/L)R (%)aMIC range (mg/L)MIC50 (mg/L)MIC90 (mg/L)R (%)a
  1. R, resistant; TMP-SXT, trimethoprim–sulphamethoxazole.

  2. aIntermediate strains were included in the resistant category.

  3. bAccording to EUCAST breakpoints.

Oxacillin≤0.06–20.251≤0.06 to >640.2545 (10.4)
Gentamicin≤0.125–414≤0.125 to >128144 (8.4)
Ciprofloxacin0.25 to >320.5427 (81.8)0.03 to >3211637 (77.1)
TMP-SXT0.016–320.1250.251 (3.0)0.023 to >320.25328 (16.7)
Tigecyclineb0.015–0.50.1250.250.015–20.0616 (12.5)

A total of 81 strains (33 normal S. aureus strains and 48 paired SCVs) were genotyped by PFGE (Fig. 3). PFGE bands of S. aureus and SCV strains with the same profile were considered to indicate clonality [15]. Whereas SCVs and their normal counterparts showed clonality in 14 of the patients, five patients exhibited different genotypes for SCV and normal strains. One patient exhibited different SCV genotypes and also different normal S. aureus genotypes in a single sample. Persistence of the same SCV was seen in five patients.

Figure 3.

 Pulsed-field gel electrophoresis patterns (SmaI digest). Lanes 1 and 2: patient 14 (small-colony variants (SCVs)). Lane 3: patient 20 (normal Staphylococcus aureus). Lanes 4 and 5: patient 20 (SCVs). Lane 6: patient 8 (normal S. aureus). Lane 7: patient 8 (SCVs). Lanes 8 and 9: patient 10 (normal S. aureus). Lanes 10, 11, 13, and 14: patient 2 (SCVs). Lane 12: patient 2 (normal S. aureus). M: molecular marker.


This is the first study to demonstrate the prevalence and phenotypic and genotypic characteristics of S. aureus SCVs in CF patients in Turkey. The prevalence of S. aureus colonization in our CF centre, which is the reference CF centre in Turkey, was 57.7% during the study period. The US Patient Registry Annual Data Report 2009 indicated that the S. aureus prevalence in CF patients was 51.3% [16]. The European CF S. aureus prevalence varies between 60.6% and 72% in different CF centres (UK, Germany, Belgium, and France) [1,17,18]. SCVs were isolated from 8.1% of CF patients and 6.4% of specimens evaluated. Among S. aureus-positive patients, the prevalence was 16.2%. In the study of Besier et al., respiratory specimens of 252 CF patients in Germany were evaluated, and the SCV prevalence was found to be 17% among S. aureus carriers, which is similar to our findings. The median age of our patient population was 9.9 years, and that of the SCV-positive patients was 14.4 years. The median age of CF patients in Besier’s study was 16 years, and of the SCV-positive patients was 21 years [9]. Kahl et al. [11] reported a high prevalence (49.1%) of SCVs in their S. aureus-positive CF population, in which the median age of SCV-positive patients was 13 years, and all SCV-positive patients received long-term trimethoprim–sulphamethoxazole prophylaxis. In one recent study, SCV prevalence was reported to be 8.2%, similar to our findings. However, the authors focused on only one respiratory specimen per patient in a short period [10]. The emergence of S. aureus SCVs has been reported to be associated with antibiotic use, but isolation after extended antibiotic-free intervals may also be possible [6,19]. The median ages of our patient population seemed to be lower than that mentioned in the studies of Besier and Kahl, and it was also determined that no trimethoprim–sulphamethoxazole prophylaxis had been applied in our CF population. The lower SCV prevalence in our study group might be attributable to these two factors.

S. aureus colonization is highest in infants and young children with CF [20]. However, the emergence of SCVs was found to be associated with advanced age, and the reason might be the increased consumption of antibiotics with increasing age [9,17]. This relationship was also observed in our study.

It is well documented that the use of MSA for the recovery of S. aureus is a prerequisite for CF microbiology [21]. In a multicentre survey from Belgium, SCV prevalence was found to be lower (4%) than in other studies; however, the authors indicated that almost all SCVs were recovered from patients in a single laboratory, which processed specimens from two centres. It was also indicated that appropriate techniques were not used in most centres, so they failed to detect any SCVs [17]. In this study, all SCVs were initially detected in MSA plates, allowing proper isolation and further identification. According to our study, the best method for detecting SCVs was to inoculate specimens onto both MSA and BHIA 5% NaCl as well as Columbia blood agar. When MSA and BHIA 5% NaCl were used together, detection and identification of 86.6% of SCVs were achieved. These selective media suppress the growth of P. aeruginosa and support the growth of normal S. aureus and SCVs [11]. Thus, MSA together with BHIA 5% NaCl could be an appropriate choice for SCV isolation, especially in laboratories with fewer facilities.

P. aeruginosa strains in CF patients produce 4-hydroxy-2-heptylquinoline-N-oxide, which suppresses the growth of S. aureus, and prolonged exposure causes the emergence of S. aureus SCVs [22–24]. Besier et al. reported that co-colonization with P. aeruginosa was significantly more frequent in patients with S. aureus SCVs than in patients with normal S. aureus only [9]. We found that 50% patients with S. aureus SCVs (10 of 20 patients) were co-colonized with P. aeruginosa, whereas only 28.56% (30 of 105) of patients with normal S. aureus were simultaneously positive for P. aeruginosa. However, the difference between the groups was not significant in our study.

Lack of biosynthesis of menadione or haemin occurs in electron transport-deficient SCVs, and this phenotype can be reversed by supplementation of media with menadione or haemin. Nonetheless, SCVs without auxotrophism for thymidine, menadione or haemin were reported to be CO2-dependent [4,25]. Auxotrophism for thymidine was detected in a small number of SCVs in our study as compared with the results of some previous studies. Auxotrophism was reported for haemin in 10 strains, for menadione in two strains and for thymidine in 41 strains among 78 S. aureus SCVs by Kahl et al. However, the selection of these auxotrophic strains was attributed to long-term trimethoprim–sulphamethoxazole prophylaxis or interventional aminoglycoside treatment [11]. In another study, thymidine auxotrophism was detected in 63% of the strains [9]. The lower number of auxotrophic strains detected in our study might be attributable to the fact that there is no antistaphylococcal prophylaxis and no predominant use of aminoglycoside treatment in cases of staphylococcal lung disease in our centre. Also, the SCV phenotype of our strains might also be attributable to CO2 auxotrophism, which we could not detect. Another possible explanation could be the reversion of some of the strains during auxotrophism testing on Mueller–Hinton agar. Besides MHA, several other media, such as chemically defined medium (CDM) or tryptic soy agar, were used in previous studies [5,11]. Further studies are needed to determine which media are best for testing auxotrophism in S. aureus SCVs.

As S. aureus SCVs grow slowly, they have decreased susceptibility to cell wall-active antibiotics [26,27]. In vitro and in vivo selection of mutants that are more resistant to ciprofloxacin among clinical S. aureus SCVs has been reported before [9,28,29]. Thus, it is not surprising that the number of strains that were resistant/intermediately resistant to ciprofloxacin was higher than the number that were resistant/intermediately resistant to the other antibiotics among normal S. aureus and SCV strains, as fluoroquinolones have been used predominantly in our study population. All of the thymidine-dependent strains that formed fried-egg colonies were resistant to trimethoprim–sulphamethoxazole in our study, in agreement with previous studies [1]. Although S. aureus SCVs were found to be more antibiotic-resistant than their simultaneously isolated counterparts, no significant difference was detected, probably because of the small number of strains tested.

The intracellular activity of tigecycline was evaluated in an infected cell model, and no higher activity was observed against SCV strains than against normal or revertant phenotypes [23]. To the best of our knowledge, our study is the first to show that MIC values for tigecyline were significantly higher in SCVs than in the simultaneously isolated normal S. aureus strains. Tigecycline resistance among S. aureus strains is very rare [13]. Consistent with the literature, we did not detect any resistance among normal S. aureus strains. However, the noteworthy finding of our study is that resistant strains are SCVs.

Genotyping by PFGE revealed clonality between S. aureus SCVs and simultaneously isolated normal S. aureus strains in a group of patients. However, some of the patients harboured different clones. When consecutive strains of patients were analysed longitudinally, it was observed that S. aureus strains in some patients could transform into different genotypes. The persistence of a predominant clone in a patient might indicate prevention of colonization by other lineages in the airways of patients, probably because of bacterial interference. Although SCVs and normal S. aureus strains have a tendency to persist in CF airways, transient/persistent colonization by another clone could be possible. Similar observations have been made for persistent P. aeruginosa colonization in CF patients [30,31].

This first study to evaluate S. aureus SCVs in CF patients in Turkey indicated the need for proper isolation, identification and reporting of S. aureus SCVs in the CF population, as the presence of S. aureus SVCs in airways will probably indicate adaptation of bacteria in airways and persistent infection. This information might have an impact on antibiotic treatment decision-making, as SCVs tend to resist intracellular killing and are more resistant to antibiotics.


We are deeply grateful to M. Hayran for his help with the statistical analysis.

Transparency Declaration

This research was supported by grants from Hacettepe University Scientific Research Foundation, Ankara, Turkey (Project no. 06 D03 101 007). We declare that we have no conflicts of interest.