In Vitro Susceptibility of Clinical Staphylococcus aureus Small-Colony Variants to β-Lactam and Non-β-Lactam Antibiotics

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Staphylococcus aureus represents one of the major invasive pathogens causing a wide range of clinically important and life-threatening infections worldwide (1). Small-colony variants (SCVs) represent a naturally occurring slow-growing subpopulation of S. aureus associated with chronic and recurrent courses of infection (2). The SCV phenotype is characterized by an altered metabolism as an adaptation to their intracellular lifestyle (3–5). β-Lactam antibiotics (BLAs), especially penicillinase-stable penicillins or first- and second-generation cephalosporins, are antimicrobials of choice for the treatment of severe S. aureus infections unless methicillin-resistant S. aureus (MRSA) is involved (6). Most of the data on the susceptibility of S. aureus SCVs toward β-lactams and other antibiotics have been derived from studies performed with deletion mutants mimicking the SCV phenotype or SCV isolates colonizing the airways of cystic fibrosis patients (7–9). However, only little is known about the effects of BLAs on S. aureus SCVs recovered from clinical samples. In this study, we investigated the susceptibilities of stable clinical SCVs and their corresponding clonally identical clinical strains with a normal phenotype (NP) toward a broad set of BLAs and non-BLAs.

(This work was presented in part at the 52nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 9 to 12 September 2012 [10].)

Ten methicillin-susceptible S. aureus (MSSA) strain pairs recovered from clinical samples (skin and soft tissue infections, bone and joint infections, wound infections, and intra-abdominal infections) at the University Hospital Münster, Germany, were included. Isolates from cystic fibrosis patients were excluded. Each strain pair consisted of a stable SCV and its corresponding NP for which clonality was confirmed by pulsed-field gel electrophoresis (11, 12). Since spontaneous switching of SCVs into the NP is possible (13), stable phenotypes were confirmed by observation of colony morphology during the experiments. Auxotrophy testing was performed as described elsewhere (9) using 10 μl of hemin (1 mg/ml), menadione (160 μg/ml), and thymidine (100 μg/ml; Sigma-Aldrich, Inc., St. Louis, MO, USA) and Mueller-Hinton agar (MHA; Merck KGaA, Darmstadt, Germany). None of the SCVs exhibited auxotrophy for the tested substances. In addition to clinical strain pairs, genetically defined stable hemB- and thyA-knockout mutants of MSSA laboratory strains 8325-4 and SH1000, respectively, and their corresponding parental and complemented mutant (CM) strains were tested (14, 15). Furthermore, MSSA reference strain ATCC 29213 was included as a quality control.

The MICs of 12 BLAs and 13 non-BLAs (Table 1) were determined by the gradient diffusion method (Etest; bioMérieux, Marcy-l'Étoile, France), according to the manufacturer's recommendations. Briefly, a bacterial suspension was prepared from colonies growing overnight on solid medium and adjusted to a turbidity of 0.5 McFarland. This inoculum was streaked onto MHA (Merck KGaA) using a plate rotator (type EPA; Robin SA, Beaucouzé, France). Etest strips were applied within 15 min after the excess moisture dried. For clinical SCVs not growing on MHA as well as for the thyA-knockout mutant, MHA with 5% sheep blood (Merck KGaA, Darmstadt, Germany) was used. The MICs were read and documented after 18 h of incubation at 36°C and for oxacillin and vancomycin after 24 h, according to the recommendations of the manufacturer. Since SCVs exhibit deficient growth, MICs were also read after 48 h of incubation. The results were read by three investigators, and median MICs were calculated for analysis. Since the appearance of single colonies in the inhibition zone was observed after prolonged incubation in the preliminary experiments, the plates were further incubated and read every 24 h for a total period of 7 days to check for appearance of colonies in the inhibition zone, indicating surviving cells.

For most tested BLAs, the MIC₅₀ and MIC₉₀ were either equal to or slightly lower for clinical SCVs than for the corresponding NPs (Table 1). The same applied to the MICs for hemB and thyA mutants compared to their wild types (Table 2). Since incubation was performed for 48 h, considerable MIC reading bias due to slower growth of SCVs is unlikely. As expected for MSSA, all strain pairs were susceptible to cefoxitin and oxacillin, with SCVs showing MICs slightly lower than those for NPs. The hemB and thyA mutants also exhibited oxacillin MICs lower than those of corresponding NPs and CMs. This is in accordance with earlier reports assessing the MICs of defined hemB mutant strains (16, 17). The cefoxitin MICs of the tested mutants were comparable to those from corresponding NPs and CMs. Interestingly, intracellular forms of menD mutant SCVs were shown to be hypersusceptible to BLAs in a recent study (18).

The MIC₅₀s of most non-BLAs were identical to or slightly lower for clinical SCVs than for NPs, whereas the MIC₉₀ values for most non-BLAs were identical or slightly higher for SCVs than for NPs. In seven of 10 pairs, the SCV strains had slightly or considerably lower MICs for gentamicin than their corresponding NP strains after 18 h of incubation (see Table S1 in the supplemental material). This finding is in contrast to those of prior reports about the reduced susceptibility of SCVs to aminoglycosides (9, 11, 19, 20) and may suggest that most clinical SCVs tested here exhibited an intact electron transport, which implies functional aminoglycoside uptake (21). However, we also found SCVs with higher gentamicin MICs than those of their NPs in two strain pairs, resulting in categorization as resistant, while the corresponding NPs were susceptible (Table S1). The hemB mutant exhibited a higher MIC for gentamicin than for their corresponding NP and CM (Table 2). This is in accordance with previous findings (14, 16, 17, 22) and is explained by electron transport deficiencies due to the hemB knockout. Interestingly, Garcia et al. postulated that gentamicin used in cell culture-based experiments remained highly bactericidal against genetically defined electron transport-deficient SCVs in spite of its increased MIC (7). For the thyA mutant, the gentamicin MICs were similar to those of corresponding NP and CM as described before for strains with mutations in thyA (23). For other tested non-BLAs, the SCV mutants exhibited no major differences from NPs and CMs irrespective of their knockouts, except for clarithromycin and clindamycin, toward which the mutants showed, as expected, resistance due to allelic replacement of hemB or thyA genes by erythromycin resistance cassette ermB (Table 2).

For some strains, we observed a regrowth phenomenon occurring as single colonies in the inhibition zone of several antibiotics. Typically, the regrown colonies appeared circularly in a concentration-dependent manner surrounding the Etest strip (Fig. 1) after prolonged incubation of up to 7 days. The regrowth was found more frequently with BLAs (19.2%) (Fig. 1 and Table 1). However, there were no marked differences between clinical NPs (17.5%) and SCVs (20.8%) regarding the occurrence of this phenomenon (Table 1). The regrowth colonies observed may result from survived persister cells. Further detailed investigations of this phenomenon are warranted, which should incorporate time-kill studies to investigate the impact of the slow growth of SCVs on killing kinetics.

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FIG 1

Example of regrown S. aureus colonies (normal phenotype) in the inhibition zone of oxacillin gradient diffusion test appearing on day 6 (just becoming visible) and day 7. Inset, 3-fold magnification of regrown colonies on day 7.

In conclusion, BLAs and other antibiotics used in this study showed high in vitro activities against a diverse set of clinical MSSA-SCVs. Thus, the detection of SCVs in clinical samples should not curtail a priori the choice of appropriate antibiotics. Clinical studies on optimal treatment regimens for SCV-associated infections are warranted.