Virulence factor expression by Gram-positive cocci exposed to subinhibitory concentrations of linezolid

Curtis G. Gemmell1,* and Charles W. Ford2

1 University Department of Bacteriology and Immunology, University of Glasgow, Royal Infirmary, 84–86 Castle Street, Glasgow G4 OSF, UK; 2 Pharmacia Corp., Kalamazoo, MI, USA

Received 12 February 2002; returned 7 June 2002; revised 15 July 2002; accepted 23 July 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Linezolid is a new oxazolidinone with potent antibacterial activity against Gram-positive cocci; it uniquely inhibits bacterial translation through inhibition of 70S initiation complex formation. The effects of sub-growth-inhibitory concentrations of linezolid on the expression of various structural and soluble virulence factors of Staphylococcus aureus and Streptococcus pyogenes were examined. For S. aureus, strains Wood 46 and Cowan 1 (NCTC 8532) were used to measure protein A, coagulase, {alpha}-haemolysin (hla) and {delta}-haemolysin (hld). For S. pyogenes, strain NCTC 9994 was used to measure M protein, streptolysin O (SLO) and DNase. Coagulase was assayed by clotting of citrated rabbit plasma, and hla, hld and SLO by lysis of rabbit, human and horse erythrocytes, respectively. Protein A and M protein were measured indirectly using bacterial susceptibility to phagocytic ingestion of radiolabelled bacteria by human neutrophils. When S. aureus was grown in 1/2, 1/4 and 1/8 MIC, linezolid, coagulase, hla and hld production were impaired. Susceptibility to phagocytosis was changed by growth in the presence of 1/2 MIC linezolid compared with that in its absence (50.8 ± 4.1% versus 38.9 ± 2.9%; P <= 0.05). When S. pyogenes was grown in 1/2, 1/4 and 1/8 MIC linezolid, SLO and DNase production were impaired compared with that of bacteria grown in the absence of the drug; its susceptibility to phagocytosis was also increased (52.8% bacteria ingested versus 37.5%; P <= 0.05). A reduction in virulence factor expression at sub-MIC linezolid concentrations may be of benefit in the treatment of Gram-positive infections.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Staphylococcus aureus and Streptococcus pyogenes are known to synthesize a variety of proteins, enzymes and toxins that are virulence factors in the infection process.1,2 Staphylococcus {alpha}-haemolysin (hla) ({alpha}-toxin) is a pore-forming toxin possessing cytolytic, haemolytic and toxic properties.3,4 S. aureus also produces a number of other virulence factors, including coagulase, other haemolysins (ß-, {gamma}- and {delta}-), leucocidin and protein A, which are likely to play a part in the infection process.57 S. pyogenes produces M protein, an inhibitor of serum opsonization, as well as streptolysin O (SLO) and streptolysin S, and deoxyribonuclease (DNase). The streptolysins display toxic activity for a variety of cell types.8,9 Both Gram-positive cocci, rich in either protein A or M protein, display resistance to phagocytosis by polymorphonuclear leucocytes, allowing bacterial survival and promoting infection.5,9 The effect of certain antibiotics on the expression of staphylococcal and streptococcal virulence factors in relation to the severity of infection and subsequent efficacy has been a topic of recent interest.6,1015

Antimicrobial agents at subinhibitory concentrations have been shown to affect the host–bacterium relationship, especially with regard to inflammatory cell function.3,11 Hitherto, the activity of antimicrobial agents has been based largely on the quantitative measurement of bacterial killing in vitro, under the assumption that achievement of specific serum concentrations above the MIC is likely to be clinically effective. However, many drugs, while not achieving their MIC for a specific pathogen in tissue, may still act in concert with inflammatory response cells.11 Clindamycin at concentrations below the MIC has been shown to impair virulence factor expression.11,16,17 Strains of S. aureus grown in the presence of as little as 1/8 MIC clindamycin still inhibited expression of {alpha}-haemolysin, {delta}-haemolysin and coagulase.18 In addition, the expression of protein A of S. aureus is altered when the organism is exposed to this antibiotic at concentrations below the MIC, leading to increased microbial susceptibility to phagocytosis and suggesting additional therapeutic efficacy.12 A recent report has shown that clindamycin inhibits differentially the transcription of certain exoprotein genes (spa and hla), probably by an effect on a regulatory protein.19

The study evaluated the effect of linezolid on the expression of structural and soluble virulence factors of S. aureus and S. pyogenes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

Two S. aureus strains, Wood 46 and Cowan 1 (NCTC 8532), and one S. pyogenes strain, NCTC 9994, were used. These strains were chosen on the basis of their ability to express virulence factors readily in vitro. Each strain was grown in the appropriate culture medium to ensure that satisfactory levels of each virulence factor were expressed. For example, S. aureus was grown in either yeast extract dialysate medium20 for {alpha}-haemolysin, or nutrient broth (Oxoid Ltd, Basingstoke, UK) for {delta}-haemolysin and coagulase. For S. pyogenes, brain–heart infusion broth (Oxoid Ltd) was used as growth medium for each of the products (SLO and DNase).

Experimental conditions

MICs of linezolid were determined by growing each bacterium for 24 h in supplemented or unsupplemented Mueller–Hinton broth (Oxoid Ltd) according to NCCLS guidelines. Thereafter, specified culture media containing 1/2, 1/4 and 1/8 MIC of linezolid for each strain were prepared, in order to measure growth and virulence factor expression in the presence of these concentrations of drug.

Measurement of bacterial toxins

Staphylococcal {alpha}- and {delta}-haemolysins. For the {alpha}-haemolysin, doubling dilutions of culture supernatant, centrifuged at 10 000g to remove bacteria, were made in diluent [phosphate-buffered saline (PBS), pH 7.1] and an equal volume of 2% v/v freshly washed rabbit erythrocytes was added. The tubes were incubated at 37°C for 60 min, at which time the highest dilution of culture supernatant causing haemolysis of 50% of the erythrocyte suspension was taken as the titre. The endpoint could be assessed accurately by measuring spectrophotometrically the release of haemoglobin against an appropriate standard curve. Haemolytic activity could be neutralized by specific antibody prepared by rabbit immunization (serum kindly donated by Dr C. Adlam, Wellcome Laboratories, UK). A similar technique was used to measure {delta}-haemolysin, except that human erythrocytes were used as target cells.

Staphylococcal coagulase. S. aureus 12009 was grown in nutrient broth with shaking for 16 h, and culture supernatant was collected by centrifugation. Doubling dilutions of culture supernatant were made in diluent comprising PBS and 10% v/v nutrient broth, and an equal volume of 1/10 v/v citrated rabbit plasma was added. Clotting of the plasma after 4 h of incubation at 37°C indicated the presence of coagulase; the titre was taken as the highest dilution to produce a measurable clot.

Streptococcal haemolysin. The O-labile haemolysin of S. pyogenes NCTC 9994 was prepared by growing the bacterium in Todd–Hewitt broth for 20 h. Culture supernatant was removed and the haemolysin stabilized by the addition of 0.1% cysteine (Sigma Ltd, Poole, UK). The haemolytic activity was measured by carrying out doubling dilutions of the supernatant in diluent also containing 0.1% cysteine, followed by the addition of 2% v/v human erythrocyte suspension. The endpoint was taken as the highest dilution of culture supernatant still able to cause lysis of 50% of the erythrocytes. Haemolytic activity could be neutralized by specific antibody produced by rabbit immunization.

Streptococcal DNase. S. pyogenes NCTC 9994 was grown for 24 h in brain–heart infusion broth. Serial dilutions of the culture supernatant were made in PBS and 20 µL aliquots were added to wells of 3 mm diameter cut in DNA agar (Oxoid Ltd). The plates were incubated for 24 h and DNase activity was measured following the addition of 1 M HCl to the plate. Clear areas around the wells denoted enzyme activity; zone sizes were measured and the endpoint was taken as the highest dilution to produce >=4 mm clearing.

Measurements of bacterial susceptibility to opsonophagocytosis

Preparation of bacteria. Bacteria were grown for 24 h in the presence or absence of [3H]adenine (for S. aureus) or [3H]thymidine (for S. pyogenes). Bacteria were harvested, washed and standardized spectrophotometrically so that suspensions containing 1 x 108 cfu/mL (bacterial ingestion studies) or 5 x 108 cfu/mL (measurement of respiratory burst) were available for investigation.

Serum opsonization. The standardized bacterial suspensions were opsonized in either 10%, 5% or 2% v/v normal human serum for 15 min at 37°C; the opsonized cells were harvested by centrifugation and resuspended in PBS at the original concentration. Unopsonized bacteria (no serum) were used for comparison.

Isolation of neutrophils (polymorphonuclear leucocytes). Polymorphonuclear leucocytes (PMNL) were isolated from human blood donations using density gradient centrifugation through Ficoll-Hypaque (Polymorphprep; Nycomed, Amersham, UK). The neutrophil-rich layer was harvested and washed gently with Hanks balanced salt solution (HBSS) containing 0.1% gelatin (gel–HBSS) before standardization to a concentration of 1 x 107 cells/mL. Purity was >=95% and viability, as measured by exclusion of Trypan Blue, was also >=95%.

Measurement of bacterial ingestion. Preopsonized radiolabelled bacteria (100 µL) were added to 100 µL of standardized suspension of PMNL in duplicate, to give a ratio of bacteria/PMNL of 10:1. The mixture was incubated for up to 30 min and the reaction stopped by the addition of 2.5 mL of scintillation fluid (EcoScint A; National Diagnostics, Atlanta, GA, USA) to one set and 2.5 mL of ice-cold (4°C) gel–HBSS to the other. This latter set was centrifuged at 1000g/5 min three times, with washing and shaking with PBS on each occasion to remove trapped or loosely adherent bacteria from the neutrophils. Finally, scintillation fluid was added to the cell pellet. Levels of radioactivity were measured in all tubes and the percentage ingestion of bacteria was calculated from the formula:

% ingestion =[(PMNL-associated radioactivity – non-specific background radioactivity)/(total radioactivity – non-specific background radioactivity)] x 100

Experiments were performed in triplicate.

Microscopic evaluation of phagocytosis. Slide preparations were made of a selection of the bacteria/PMNL mixtures, as follows. After the washing stages described for measurement of phagocytic ingestion, the reaction mixture was resuspended in 50 µL of PBS and centrifuged at 500 rpm for 5 min on to silane-coated glass slides, using a Cytotek centrifuge (Miles Scientific, Eckhardt, IN, USA). After drying for 10 min, the slides were stained with Giemsa stain and examined microscopically. The percentage of PMNL showing ingestion of bacteria, as well as the average number of bacteria per phagocytic cell, were calculated. Experiments were performed in triplicate.

Measurement of respiratory burst. The phagocytic process was also measured using chemiluminescence to follow the respiratory burst in neutrophils exposed to serum-opsonized bacteria. Opsonized bacteria (5 x 107 cfu) were added to a reaction mixture consisting of 5 x 105 PMNL and 50 µL of 1 x 10–5 M luminol in the dark. Light release was then measured over a 30 min period and the peak response (in mV) noted. Experiments were performed in triplicate.

Statistics

Comparisons between the effect of 1/2, 1/4 and 1/8 MIC of linezolid exposure and growth in the absence of drug for the quantification of bacterial toxins, including staphylococcal {alpha}- and {delta}-haemolysins, measurements of bacterial susceptibility to opsonophagocytosis, measurements of bacterial ingestion using the percentage of PMNL showing ingestion of radiolabelled bacteria, and measurement of respiratory burst using peak chemiluminescence response, were made using analysis of variance (ANOVA) and t-tests, as appropriate. All statistical tests were two-sided, and P values <=0.05 were considered statistically significant. All analyses were performed using Excel (Microsoft, Redmond, WA, USA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility of bacterial strains to linezolid

The in vitro susceptibility of both S. aureus strains (MIC 1.5 mg/L) and S. pyogenes NCTC 9994 (MIC 0.5 mg/L) was consistent with those described previously for linezolid.24

Effect of sub-MIC linezolid exposure on toxin production

Linezolid inhibited {alpha}-haemolysin, {delta}-haemolysin and coagulase production by S. aureus, when exposed to 1/2, 1/4 and 1/8 MIC linezolid compared with that in the absence of any drug (Table 1). These differences in toxin expression can be attributed directly to an action of the drug on protein synthesis, since growth curves of S. aureus in the presence of 1/2 and 1/4 MIC linezolid were similar to those in the absence of the drug. Likewise, linezolid significantly impaired SLO and DNase production by S. pyogenes strain NCTC 9994 when exposed to 1/2, 1/4 and 1/8 MIC linezolid (Table 2). In each case, toxin yield was measured after 24 h of growth in the presence or absence of linezolid. Although the kinetics of growth were unchanged, the kinetics of antibiotic impairment of toxin production remain to be investigated.


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Table 1.  Effect of linezolid at subinhibitory concentrations on virulence factor expression by S. aureus
 

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Table 2. Effect of linezolid at subinhibitory concentrations on virulence factor expression by S. pyogenes NCTC 9994
 
Effect of sub-MIC linezolid exposure on susceptibility of S. aureus and S. pyogenes to phagocytosis

Modest potentiation of opsonophagocytosis was demonstrated in S. aureus (Cowan 1) grown in the presence of subinhibitory concentrations of linezolid (Figure 1). The greatest effect was seen using 5% normal human serum as opsonic source (P < 0.05). Similar changes were also observed in stained preparations of the bacterium/PMNL mixture. More linezolid-grown S. aureus were observed intracellularly as compared with the same bacteria grown in the absence of the drug. Significant potentiation of opsonophagocytosis was demonstrated in S. pyogenes (NCTC 9994) grown in the presence of sub-MIC linezolid (Figure 2). This effect was seen when either 10% or 5% normal human serum was used as opsonin. Visualization of the bacterium/phagocyte mixture using light microscopy revealed differences between the appearance of PMNL ingesting linezolid-treated (1/2 MIC) S. pyogenes; greater numbers of intracellular bacteria were observed (Figure 3) compared with PMNL exposed to streptococci grown without the drug (Figure 4). A quantitative measurement of PMNL-ingested bacteria (phagocytic index) also revealed modest differences (P > 0.05) between S. pyogenes grown in the presence or absence of linezolid (Figure 5). However, many more bacteria were seen intracellularly when linezolid-grown staphylococci were incubated with PMNL than were seen with untreated bacteria (P < 0.05).



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Figure 1. Phagocytic ingestion of S. aureus Cowan 1 strain grown in the presence and absence of linezolid.

 


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Figure 2. Phagocytic ingestion of S. pyogenes NCTC 9994 strain grown in the presence or absence of linezolid.

 


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Figure 3. Microscopic appearance of phagocytic ingestion of linezolid-grown S. pyogenes NCTC 9994. Magnification ¥400.

 


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Figure 4. Microscopic appearance of phagocytic ingestion of S. pyogenes NCTC 9994 grown in the absence of linezolid. Magnification ¥400.

 


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Figure 5. Cytological measurement of ingestion of S. aureus Cowan 1 and S. pyogenes NCTC 9994 by PMNL following growth in the presence or absence of sub-MIC linezolid.

 
Respiratory burst

The induction of a respiratory burst by PMNL following exposure to S. aureus (Cowan 1) grown in the presence or absence of linezolid at sub-MIC levels was measured as the maximal chemiluminescence (mV) response, usually occurring 15–20 min after addition of PMNL to the bacterium/luminol reaction mixture in the dark. The maximal chemiluminescence responses of PMNL to bacteria grown in the absence of the drug, compared with those grown in the presence of 1/2 MIC linezolid, 1/4 MIC linezolid and 1/8 MIC linezolid in 10% serum, were 27.8, 29.1, 35.8 and 35.8 mV, and in 0% serum these values were 20.6, 26.5, 28.3 and 28.1 mV, respectively. The level of respiratory burst of PMNL exposed to opsonized or unopsonized bacteria was not statistically different in untreated versus drug-treated bacteria.

For S. pyogenes NCTC 9994, bacteria were exposed to 1/2 MIC linezolid or no drug and opsonized with either 10% or 0% serum. Following opsonization, maximal chemiluminescence (mV) responses of PMNL were 35.6 and 21.8 mV to bacteria grown in the absence of the drug, and 49.0 mV and 20.5 mV to bacteria grown in the presence of 1/2 MIC linezolid, in 10% and 0% serum, respectively (Figure 6). Half-MIC linezolid-grown streptococci stimulated a significantly higher maximal chemiluminescence response compared with that observed in the absence of the drug (P < 0.05).



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Figure 6. Maximal chemiluminescence (mV) following exposure of PMNL to S. pyogenes grown in the presence or absence of sub-MIC linezolid.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sub-MIC concentrations of linezolid inhibited virulence factor expression, as demonstrated by a significant decrease in toxin and enzyme production by S. aureus and S. pyogenes. Exposure to linezolid at concentrations below the MIC potentiated susceptibility of S. aureus and S. pyogenes to opsonophagocytosis by human neutrophils. These results were observed at concentrations that did not inhibit bacterial growth.

Ohlsen et al.4 evaluated the effects of 31 antibiotics on the expression of {alpha}-toxin in methicillin-sensitive and methicillin-resistant S. aureus isolates at concentrations below the MIC, using a gene fusion model, and demonstrated that subinhibitory concentrations of some antibiotics altered the expression of the {alpha}-toxin gene. The most dramatic observation was the almost complete inhibition of {alpha}-toxin expression by clindamycin and the strong induction of {alpha}-toxin expression by ß-lactam antibiotics. Glycopeptide antibiotics had virtually no effect on {alpha}-toxin expression; erythromycin and the aminoglycosides reduced expression. It was suggested that reduced virulence factor expression in Gram-positive bacteria in the presence of some antibiotics may be relevant to the clinical management of Gram-positive infections.4 Indeed, there has been speculation that the addition of clindamycin to standard penicillin therapy caused more rapid resolution of invasive streptococcal infection and toxic shock syndrome.21 Therefore, in therapy, additional significance may be attached to drugs such as clindamycin and, as shown in this study, linezolid. Specific examples relating this phenomenon in the clinical setting are rare. However, several animal models have investigated the role of adhesins, haemolysins and enterotoxins produced by S. aureus in the development of septic arthritis.6,13,22 Results of these studies suggested that {alpha}-haemolysin, {gamma}-haemolysin and protein A are important virulence factors responsible for the development of S. aureus septic arthritis.6,13

The increasing incidence of bacterial infections due to staphylococci, and the rise in methicillin- and vancomycin-resistance therein, is of great concern.1,23,24 As the number of therapeutic options is reduced as resistance to traditional antibiotics rises, modulation of virulence factor expression by antibiotic treatment may be of increasing importance. It is clear that virulence factor expression is altered by certain environmental stimuli and may contribute to the in vivo virulence of S. aureus and S. pyogenes.3 Linezolid appears to inhibit bacterial virulence factor expression at low concentrations, which might thereby contribute to its pharmacodynamic effect in the treatment of serious Gram-positive infections. Linezolid has been shown to be as effective as oxacillin–dicloxacillin in the treatment of complicated skin and soft-tissue infections. In one study,25 clinical cure rates and microbiological success rates for linezolid-treated patients were high (88.6% and 88.1%, respectively). The efficacy of linezolid may be due, in part, to the high concentrations achieved in skin, as well as the more subtle action of inhibition of toxin and enzyme production by S. aureus and S. pyogenes described in this study and in other studies with other antibiotics.8,26,27


    Acknowledgements
 
This study has been supported by a grant from Pharmacia Corporation, Kalamazoo, MI. The results were presented in part at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), San Francisco, CA, USA, 26–29 September 1999.


    Footnotes
 
* Corresponding author. Tel: +44-141-211-4654; Fax: +44-141-552-1524; E-mail: cgg1g{at}clinmed.gla.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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