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FEMS Immunology and Medical Microbiology, 37, 2003, pp. 45-51

Apotransferrin administration  prevents growth  of Staphylococcus epidermidis in serum  of stem cell transplant patients  by binding of free iron

Leni von Bonsdorff, Leila Sahlstedt, Freja Ebeling, Tapani Ruutu, Jaakko Parkkinen
 

ABSTRACT

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10³ cfu ml⁻¹ but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.

Author Keywords: Apotransferrin; Staphylococcus epidermidis; Non-transferrin-bound iron; Transferrin saturation; Stem cell transplantation; Opportunistic bacterium

1. INTRODUCTION

Infections cause significant morbidity and mortality in haematological patients receiving myeloablative conditioning therapy and allogeneic stem cell transplantation [1 and 2]. Staphylococcus epidermidis is a major causative agent of bacteraemia and septic infections in neutropenic patients receiving intensive immunosuppressive therapy [3 and 4].

During chemotherapy-related neutropenia, plasma-based resistance factors against bacterial infections may become critical. One of the factors known to influence microbial growth is iron accessibility [5]. In normal conditions, plasma iron is tightly bound to the high-affinity iron carrier protein transferrin, and the level of redox-active, non-transferrin-bound iron (NTBI) in plasma is very low. This is considered to prevent the growth of opportunistic pathogens, which do not possess efficient molecular mechanisms to utilise transferrin-bound iron [6]. On the other hand, several studies have indicated that high-dose chemotherapy of haematological malignancies is associated with an increase in serum total iron content, which often exceeds the iron binding capacity of transferrin and results in the appearance of NTBI in the serum of the patients [7, 8, 9 and 10]. Complete transferrin saturation and presence of NTBI are particularly common during myeloablative therapy and stem cell transplantation [11, 12 and 13].

There is both experimental and clinical evidence suggesting that hyperferraemia predisposes patients to infections with opportunistic pathogens. In experimental animal infections, the virulence of several organisms was markedly enhanced by iron administration to the host [14]. Several studies have found an association between fungal infections and a high transferrin saturation level in neutropenic leukaemia patients [15, 16, 17 and 18]. It has also been suggested that high transferrin saturation contributes to lethality of pneumococcal pneumonia [19]. However, it is not known whether high transferrin saturation as such, or the presence of NTBI, promotes microbial virulence.

In the present study we investigated the influence of transferrin saturation and NTBI on the growth of S. epidermidis in serum. The role of iron uptake mechanisms of S. epidermidis in the serum milieu has been unclear. Early studies indicated that in contrast to Staphylococcus aureus, coagulase-negative staphylococci were not able to grow in normal serum without iron supplementation [20]. On the other hand, it has been demonstrated that both S. epidermidis cells and the siderophore staphyloferrin A can displace iron from transferrin under in vitro conditions [21]. In our earlier study in serum-free conditions, S. epidermidis was not able to utilise transferrin-bound iron for growth unless high bacterial densities were present [22]. In the present study we found that S. epidermidis was not able to grow in serum unless either NTBI or a high initial count of bacteria was present. Further, we demonstrated that it is possible to prevent both the appearance of NTBI and growth of S. epidermidis in the serum of stem cell transplant patients by giving repeated infusions of exogenous apotransferrin.

2. MATERIALS AND METHODS

2.1. Patients

Twenty-six patients with haematological malignancies receiving allogeneic stem cell transplantation after myeloablative conditioning with cyclophosphamide and total body irradiation were enrolled in clinical trials with different doses of an investigational human plasma apotransferrin product (Apotransferrin SPR, Finnish Red Cross Blood Transfusion Service) [23]. Six patients received a single intravenous dose of 100 mg kg⁻¹ of apotransferrin on day 3 after the stem cell transplantation [24]. Twenty patients were given repeated doses daily or every second day starting 6 days before the stem cell transplantation and continuing for 14–18 days. The total dose per patient varied between 0.3 and 0.6 g kg⁻¹ in a low-dose group of 12 patients and was 1 g kg⁻¹ in a high-dose group of eight patients. Ten patients were from a previous follow-up study and did not receive apotransferrin [13]. Prophylactic antibacterial agents routinely given to the patients were sulfamethoxazole/trimethoprim or ciprofloxacin. The clinical trials were approved by the ethical committee of the Helsinki University Central Hospital. Informed written consent of the patients was obtained.

2.2. Bacterial strains and culture conditions

Two multi-resistant clinical isolates of S. epidermidis (16779 and 19435) from neutropenic patients, which were resistant to methicillin and at least two antibiotic classes other than -lactams, and the ATCC 12228 strain were used. Siderophore production was induced by precultivation in iron-depleted RPMI 1640 medium as previously described [22]. The bacteria were harvested in the late exponential growth phase and 10-fold dilutions were prepared in physiological saline.

An inoculum of 50 l was added to 200 l of serum buffered with 50 mM HEPES buffer, pH 7.4, in microwells. The growth was monitored by optical density (OD) for 24 h at 37°C with periodic shaking (Bioscreen C Microbiology Reader, Labsystems, Finland) in an air-tight chamber maintained in a 5% CO₂ atmosphere. Serum pH was maintained at 7.3–7.4 and bicarbonate level at about 17 mM throughout the culture period. The detection limit for the increase in OD in 24 h (ΔOD) was 0.05 and the threshold density of S. epidermidis which could be detected was about 10⁶ cfu ml⁻¹. Viable counts were determined by plating on trypticase-soy agar plates (bioMérieux, France). Sera with in vitro additions of ferric nitrilotriacetic acid prepared as described in [22] or apotransferrin were preincubated for 30 min at 37°C in 3% CO₂ to ensure binding of iron to transferrin.

2.3. Transferrin, iron and other serum assays

Serum transferrin (reference range 1.75–3.13 g l⁻¹) and C-reactive protein (CRP) (reference range <10 mg l⁻¹) were measured by immunoturbidimetric methods. Serum total iron was measured by a colorimetric ferene-S method (reference range 8–30 M for females and 10–31 M for males). All above-mentioned measurements were done at the HUCH Laboratory Diagnostics of the Helsinki University Central Hospital. Transferrin saturation (%) was calculated by the formula: serum iron (M)/serum transferrin (g l⁻¹)×3.98. NTBI was measured using the bleomycin-detectable iron assay adopted for small (25 l) serum volumes [25]. Briefly, bleomycin was added to serum in the presence of ascorbic acid. Redox-active iron bound to bleomycin caused degradation of DNA in the mixture and the DNA degradation products were measured in microwell plates by a colorimetric thiobarbituric acid assay. A standard curve with 0.1–3 M pure iron in water was calculated by linear regression of logarithmic values. The assay had a detection limit of 0.1 M. The pH and bicarbonate concentrations in serum were measured using a blood gas analyser (ABL700, Radiometer).

3. RESULTS

3.1. Influence of NTBI on the growth of S. epidermidis in serum

No growth of S. epidermidis took place in normal serum samples inoculated with 10³ cfu ml⁻¹ of the bacteria, whereas the bacteria proliferated rapidly in serum supplemented with iron (Fig. 1A). The three studied strains displayed similar iron dependence in their growth. Normal serum samples had transferrin saturation of 24–30% and NTBI <0.1 M. The amount of iron added to serum (45 M) was sufficient to fully saturate transferrin and form NTBI in a concentration of 3.2±0.8 M (mean±S.D., n=5). When both apotransferrin (2 g l⁻¹) and iron were added to the serum, no growth of the bacteria was observed (data not shown).

 

 

Fig. 1. Effect of in vitro addition of iron and transferrin on growth of S. epidermidis in serum. An inoculum of 10³ cfu ml⁻¹ was added to buffered serum, growth was carried out at 37°C in a 5% CO₂ atmosphere and monitored by optical density. Viable counts determined after 24 h are shown beside the growth curves. A: Two clinical isolates, 16779 and 19435, and the ATCC 12228 strain were grown in normal serum (closed symbols) and in the same serum with added NTBI (open symbols). B: Growth of strain 16779 in a patient serum containing 0.4 M NTBI and in the same serum after addition of 0.1 g l⁻¹ iron-free apotransferrin or 0.1 g l⁻¹ iron-saturated holotransferrin.

 

 

The influence of NTBI on bacterial growth in patient serum was studied with the two multiple drug-resistant S. epidermidis strains. Both strains grew rapidly in the serum samples of the three patients studied, which all contained fully saturated transferrin and NTBI (0.3±0.1 M). The addition of 0.1 g l⁻¹ apotransferrin, which bound the NTBI, fully prevented the growth of the staphylococci, whereas the addition of iron-saturated holotransferrin had no effect. The growth curves from one patient with and without transferrin additions are shown in Fig. 1B.

The influence of initial bacterial density on iron dependence of bacterial growth was studied in normal serum with and without iron addition. In iron-supplemented serum, inoculations in the range of 10²–10⁵ cfu ml⁻¹ resulted in clearly detectable growth curves by OD monitoring, whereas without iron addition only the highest inoculum resulted in detectable growth (Fig. 2A). Determination of viable counts after 24 h indicated growth to 10⁶ cfu ml⁻¹ with starting densities of 10⁴–10⁵ cfu ml⁻¹ and no detectable growth with 10²–10³ cfu ml⁻¹. After iron addition the growth consistently reached high concentrations (10⁷–10⁸ cfu ml⁻¹) with all inoculations (Fig. 2B).

 

 

Fig. 2. Effect of the initial bacterial count on the growth of S. epidermidis in normal serum and in serum containing NTBI. A: Growth curves of different initial bacterial densities in normal serum (closed symbols) and in the same serum with added NTBI (open symbols). B: Viable counts at the beginning and after 24 h in the same sera. Growth of strain 16779 was monitored as described in Fig. 1. Bars indicate the mean and S.E.M. of repeated (n=4) experiments. The difference between the viable counts after 24 h in normal serum and in the same serum with NTBI was statistically significant at all starting cell densities (10³ cfu ml⁻¹ P<0.0001, 10⁴ cfu ml⁻¹ P<0.01 and 10⁵ cfu ml⁻¹ P<0.05, Student’s t-test).

 

 

3.2. Influence of apotransferrin administration on bacterial growth in patient serum

A bacterial inoculum of 10³ cfu ml⁻¹ was used in the microwell system with ΔOD measurement, which allowed identification of samples with growth to at least 10⁶ cfu ml⁻¹>80% (Fig. 3). Growth was detected in 87% (54/62) of the samples with NTBI, whereas only 4% (3/70) of the samples without NTBI grew (P<0.0001, Fisher’>80%. Of the eight NTBI-positive samples not supporting growth, two were from a patient who had received vancomycin and four from a patient who was treated with a combination of tobramycin and ceftazidime.

 

 

Fig. 3. Relation of bacterial growth measured as an increase in optical density (ΔOD) to transferrin saturation in the serum samples of the stem cell transplantation patients (n=132). The growth of an inoculum of 10³ cfu ml⁻¹ of strain 16779 was monitored as the increase in optical density during 24 h. The line indicates the detection limit.

 

 

In the first clinical trial in which a single intravenous dose of apotransferrin was given to the patients, serum samples from all six patients were positive for NTBI before apotransferrin administration and the inoculated bacteria grew in all of them (Fig. 4). Immediately after the administration, the growth was prevented concurrently with a drop in transferrin saturation and disappearance of NTBI. The length of this bacteriostatic effect of the single apotransferrin dose varied from 12 h to several days. The bacteriostatic effect typically disappeared simultaneously with the reappearance of NTBI in serum (Fig. 4). In the serum of a patient who received vancomycin on days 8–11, no growth of the staphylococci took place in spite of high transferrin saturation and NTBI on days 10–11 ( Fig. 4B).

 

 

Fig. 4. Effect of a single intravenous administration of apotransferrin on transferrin saturation, NTBI and growth of S. epidermidis in the serum samples of three stem cell transplantation patients. Bacterial growth was measured as in Fig. 3. The arrow indicates the intravenous injection of 100 mg kg⁻¹ apotransferrin 3 days after the stem cell transplantation.

 

 

In subsequent trials, apotransferrin was administered as repeated doses starting 6 days before the stem cell transplantation on the same day as the conditioning therapy. For comparison, growth was monitored in two patients who did not receive apotransferrin (Fig. 5A). A sharp rise in serum iron content and appearance of NTBI occurred after the start of the conditioning and concomitantly, growth of inoculated staphylococci was detected. The lower apotransferrin doses tested were not sufficient to prevent the appearance of NTBI. The transferrin saturation remained at a high level, and bacteria grew in the serum samples which were positive for NTBI (Fig. 5B). Repeated higher doses prevented the appearance of NTBI in five of the eight patients studied. No significant bacterial growth could be detected in the serum of a patient with no detectable NTBI during the whole study period ( Fig. 5C).

 

 

Fig. 5. Transferrin saturation, NTBI and bacterial growth in the serum of three stem cell transplantation patients, of whom one (A) did not receive apotransferrin and two (B,C) received repeated intravenous apotransferrin infusions at two different dose levels. Bacterial growth was monitored as in Fig. 3. Arrows indicate the apotransferrin infusions, closed and open arrows corresponding to doses of 100–115 mg kg⁻¹ and 26 mg kg⁻¹, respectively.

 

 

3.3. Infection markers in the patients receiving apotransferrin

To gain insight into whether NTBI is a risk factor for septic infections, markers for septic infections (fever >38°>30 mg l⁻¹) were compared between the 10 patients who did not receive apotransferrin, 12 patients who received repeated lower doses of apotransferrin and eight patients who received repeated higher doses. Positive blood cultures were obtained only from a minority of patients with suspected septic infections, which probably was due to the use of prophylactic antibiotics and rapid onset of treatment with intravenous antibiotics when signs of septic infections were detected.

Most patients were positive for the infection markers at least on one occasion during the stem cell transplantation period. When the total number of days with positive infection markers was evaluated as a proportion of all study days, there was a significant reduction in the number of days with fever and days with elevated CRP in the patient groups who received low and high doses of apotransferrin, respectively (Table 1). When both markers were evaluated together, there was a significant reduction in the high-dose group (P<0.01). The number of days with intravenous antibacterial treatment was significantly lower in both patient groups receiving apotransferrin than in the control group. There was also a trend towards a lower proportion of days with antifungal therapy in the higher-dose group but this was not statistically significant.

 

 

Table 1. Comparison of total number of days with positive infection markers and treatment with intravenous antibiotics between the patient groups who received no apotransferrin or received increasing total doses
 

 

 

4. DISCUSSION

The results of the present study show that at initial densities up to 10³ cfu ml⁻¹, S. epidermidis was able to grow in serum only when the transferrin was fully saturated and NTBI was present. This was shown both by adding iron to normal serum and by studying serum samples of patients undergoing stem cell transplantation, in which transferrin became fully saturated and NTBI appeared after the initiation of the myeloablative therapy. Further, the growth of S. epidermidis in serum was effectively prevented when apotransferrin was added to the serum in vitro or when apotransferrin was intravenously administered to the patients to bind NTBI in circulation.

The results are in agreement with earlier findings [20], indicating that in contrast to S. aureus, coagulase-negative staphylococci could not grow in serum without iron supplementation. We conducted the bacterial culturing under conditions which kept serum bicarbonate and pH at physiological levels, both of which influence transferrin iron binding [26]. We found 80% to be a threshold for transferrin saturation, above which bacterial growth took place in patient samples. In a previous study, this was also identified as the critical saturation level, above which redox-active NTBI occurred in patients’ sera [25]. In accordance, we observed a strong association between bacterial growth and NTBI in the patient serum samples.

The ability of S. epidermidis to multiply in serum in the absence of NTBI was dependent on the initial bacterial density. The bacteria grew in normal serum only at high initial densities (10⁴–10⁵ cfu ml⁻¹) and the growth was much slower than in the presence of NTBI. This is in line with our earlier findings in serum-free conditions showing that in the presence of purified transferrin the growth of S. epidermidis was critically dependent on full transferrin saturation and the presence of NTBI [22]>10⁵ cfu ml⁻¹). It has previously been demonstrated that S. epidermidis cells at high densities (10⁸ cfu ml⁻¹) could displace iron from transferrin [21]. This was attributed to a cell wall transferrin binding protein, identified as glyceraldehyde-3-phosphate dehydrogenase. However, a more recent study on iron acquisition mechanisms of S. aureus showed that this glycolytic enzyme has no affinity for transferrin and another cell wall protein with iron-regulated expression was identified as a transferrin binding protein [27]. It is possible that multiple mechanisms, such as acidification and accumulation of chelating agents as a result of bacterial metabolism promote iron liberation from transferrin in the presence of high bacterial densities [22 and 27].

The number of invading bacteria evidently plays a role in the pathogenesis of septic infections. S. epidermidis often contaminates central venous catheters, where biofilm formation promotes the adherence and protection of the bacteria [28]. In stem cell transplant patients, the oral mucosa is another possible route of infection due to treatment-related mucositis [29]. In catheter infections, there is an association between the number of organisms retrieved from the catheter surface and the risk of infection, and infection occurs only when the number exceeds a certain quantitative threshold [30 and 31]. In a study of catheter-related bacteraemias, blood drawn from the catheters had up to 10⁵ cfu ml⁻¹ of coagulase-negative staphylococci, whereas peripheral blood samples had 10¹–10² cfu ml⁻¹ [32]. Another study showed that the mean bacterial cell concentration in peripheral blood samples in blood stream infections caused by coagulase-negative staphylococci was 33 cfu ml⁻¹ [33]. It may be reasoned that a high local density of S. epidermidis in the intravenous catheters promotes bacterial growth also in the absence of NTBI, whereas the proliferation of low counts occurring in the peripheral blood may depend on the availability of NTBI.

In the present study, apotransferrin administration to the patients effectively bound NTBI and restored the growth-inhibitory effect of patient serum against S. epidermidis. Repeated higher doses of apotransferrin maintained the growth-inhibitory effect in the serum of five out of the eight patients during the whole stem cell transplantation period. Earlier studies have described the reduced ability of serum from leukaemia patients to prevent growth of Pseudomonas aeruginosa [34], Escherichia coli, S. aureus [35] and Candida albicans [15]. Several studies have also shown that transferrin displays a growth-inhibitory effect in vitro on opportunistic organisms, such as Vibrio vulnificus [36], Rhizopus [37], C. albicans [15 and 38] and Histoplasma capsulatum [39]. Recently, an anticryptococcal protein in serum was identified as apotransferrin [40]. Thus, apotransferrin administration could have an impact on infections caused by several bacteria or fungi.

Among the small number of patients in the present study, the incidence of septic infections was not significantly different in the patients who received apotransferrin than in the control patients. When the patient groups were compared with respect to the total number of days with fever or elevated CRP, the patients who received repeated doses of apotransferrin had significantly fewer positive days than the patients who did not receive apotransferrin. Correspondingly, the number of days with intravenous antibiotics was significantly smaller during the study period in the patients who received repeated apotransferrin infusions. Although the patients were not compared in a randomised setting, this may suggest that repeated infusions of apotransferrin could protect the neutropenic patients against septic infections.

In conclusion, our results suggest a major pathogenic role for NTBI in septic S. epidermidis infections in neutropenic patients. With increasing resistance of microbes to antibiotics, binding of NTBI with apotransferrin could offer a method to enhance physiological resistance against infections. However, the clinical efficacy of this therapeutic concept remains to be investigated in prospective controlled trials.

ACKNOWLEDGEMENTS

We thank Mrs S. Lehmonen, Mrs A. Remes and Mrs T. Kupari for expert technical assistance.

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