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Human recombinant lactoferrin acts synergistically with antimicrobials commonly used in neonatal practice against coagulase-negative staphylococci and Candida albicans causing neonatal sepsis

¹ Baylor College of Medicine & Texas Children's Hospital, 6621 Fannin, MC: WT 6-104, Houston, TX 77030, USA

² Baylor College of Medicine, 1102 Bates Avenue, MC: BCM 320, Houston, TX 77030, USA

Correspondence
Mohan Pammi Venkatesh
mohanv{at}bcm.edu

Received 12 February 2008
Accepted 24 April 2008

Abbreviations: AMB, amphotericin B; CI, combination index; CoNS, coagulase-negative staphylococci; DRI, dose reduction index; FLC, fluconazole; NAF, nafcillin; TLF, human recombinant lactoferrin (talactoferrin alpha); VAN, vancomycin.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Sepsis is a major cause of death in the neonatal period, especially in preterm and very-low-birth-weight neonates (birth weight <1500 g) (Kaufman & Fairchild, 2004; Lawn et al., 2006). Sepsis significantly increases morbidity, prolongs the need for ventilation and intravascular access, increases the length of hospital stay and increases the incidence of bronchopulmonary dysplasia, necrotizing enterocolitis and adverse neurodevelopmental outcomes (Adams-Chapman & Stoll, 2006; Stoll et al., 2002, 2004). Coagulase-negative staphylococci (CoNS) and Candida albicans are among the most common organisms isolated from neonatal sepsis (Stoll et al., 2002). The global emergence of antibiotic resistance (Levy, 1998, 2001) and immature neonatal host defences dictate the need to use non-antibiotic agents that can enhance host immunity and can be used against neonatal sepsis. Lactoferrin, a component of innate immunity, is one such agent.

Lactoferrin is an 80 kDa, monomeric, diferric, cationic glycoprotein with an isoelectric point of between 8.4 and 9.0 (Moguilevsky et al., 1985; Sanchez et al., 1992) and belongs to the transferrin family of iron-binding glycoproteins. Lactoferrin is composed of 690 aa with a highly conserved three-dimensional structure, and its role in several pathophysiological functions including iron homeostasis, organ morphogenesis, host defence against infection, inflammation and cancer is emerging (Baker et al., 1998; Ward et al., 2005).

Lactoferrin is found on mucosal surfaces, in significant concentrations in human colostrum (7 g l^–1) and less so in mature human milk (1 g l^–1), tears (3.8 g l^–1), saliva (20 mg l^–1), seminal fluid and secondary granules of neutrophils (Hennart et al., 1991; Masson et al., 1966, 1969; Masson & Heremans, 1971). Normal serum levels in humans range from 0.4 to 2 mg l^–1 and may increase to 200 mg l^–1 following its release from neutrophils during sepsis (Bennett & Kokocinski, 1978). The expression and secretion of lactoferrin in significant concentrations on mucosal surfaces and its release at inflammatory sites by polymorphonuclear neutrophils have established its role as an innate immunity agent, as well as a contributor to acquired cellular and humoral immune responses.

Lactoferrin has broad-spectrum antimicrobial activity against bacteria, fungi, viruses and protozoa. Its antimicrobial activity stems from two distinct effects: (i) its iron-sequestering ability, which can be negated by saturation with iron (Kalmar & Arnold, 1988; Yamauchi et al., 1993); and (ii) its iron-independent killing due to a direct interaction with the microbial surface resulting in cell lysis (Farnaud & Evans, 2003; Orsi, 2004; Valenti & Antonini, 2005).

Bovine lactoferrin has been shown to inhibit planktonic (free-living) organisms, as well as biofilms of Staphylococcus epidermidis growing on soft contact lenses in vitro (Leitch & Willcox, 1999b). Both bovine and human lactoferrins inhibit Candida isolates from the oral cavity of immunocompromised patients by their action on Candida cell membranes (Xu et al., 1999) and exhibit synergy with common antifungal agents (Kuipers et al., 1999). Synthetic lactoferrin-derived peptides have been shown to have a greater antifungal effect compared with native lactoferrin in vitro, and the first two arginines at the N-terminus of human lactoferrin appear to be critical in the candidacidal activity (Lupetti et al., 2000).

Synergistic effects of lactoferrin with antimicrobial agents against S. epidermidis and Candida species have been reported (Leitch & Willcox, 1999b; Lupetti et al., 2003), but the term synergy in drug combinations has been loosely applied without definite modelling or derivations, and has not been evaluated at multiple dose effects or drug-dose ratios (Odds, 2003). The utility of combining human recombinant lactoferrin (talactoferrin alpha; TLF) with the commonly used antimicrobials in neonatal practice to enhance antimicrobicidal effects has not yet been reported.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Antimicrobials. TLF expressed in Aspergillus awamori (Ward et al., 1995) was provided by Agennix. TLF stock solution (100 mg ml^–1) was diluted to give serial twofold dilutions starting from a concentration of 8300 µg ml^–1. Fluconazole (FLC) stock solution (2 mg ml^–1; Sigma-Aldrich) was diluted to give serial twofold dilutions starting from 64 µg ml^–1. Amphotericin B (AMB) obtained from Streptomyces (Sigma-Aldrich), vancomycin (VAN; USB) and nafcillin (NAF; Sigma-Aldrich) were used in serial twofold dilutions starting from 4 µg ml^–1.

Organisms. The organisms used were S. epidermidis ATCC 55133, seven clinical strains of CoNS (S. epidermidis 100H and 101S, CoNS 102R, 103A, 104O, 105D and 106F) and C. albicans strains ATCC 32354, 200C and 201M. All clinical strains (except for S. epidermidis ATCC 55133 and C. albicans ATCC 32354, which are human non-neonatal isolates) were isolated from the peripheral blood of neonates with sepsis at Texas Children's Hospital, Houston, TX, USA.

Growth media. Sabouraud dextrose agar plates and glucose/yeast extract/peptone broth (GYEP) were used for Candida subcultures, and RPMI 1640 without bicarbonate buffered with 0.165 M MOPS to a pH of 7 was used for antimicrobial susceptibility testing. For CoNS, trypticase soy agar with 5 % sheep blood and trypticase soy broth (TSB) were used for subcultures and antimicrobial susceptibility testing, respectively.

Inoculum preparation. CoNS and C. albicans were plated overnight from cryogenic stocks stored at –80 °C. Five colonies were picked and inoculated into TSB or GYEP broth, respectively (75 ml in a 250 ml flask), and incubated for up to 2 h in an incubator shaker at 37 °C, resulting in growth-phase organisms. The suspension was centrifuged at 1932 g for 10 min three times and the sediment was suspended in normal saline to an OD₆₃₀ of 0.6 (corresponding to 0.5x10⁸–1x10⁸ c.f.u. ml^–1) for CoNS and an OD₄₉₀ of 0.6 (corresponding to 1x10⁷–5x10⁷ c.f.u. ml^–1) for C. albicans. Quantitative cultures of the final suspension confirmed the final concentrations.

Antimicrobial susceptibility testing of CoNS and C. albicans

MICs of TLF, NAF and VAN against CoNS. MIC₅₀ and MIC₉₀ values were determined using the broth microdilution method as recommended by the CLSI (2003, 2007) with minor modifications. Five microlitres of a 1 : 10-diluted inoculum was added to 100 µl of the serial dilutions of the antimicrobial to be tested in a 96-well microtitre plate. The final concentration of CoNS in each well of the microtitre plate was 2.5x10⁵–5x10⁵ c.f.u. ml^–1. Microtitre plates were incubated for 24 h at 35 °C. Drug-free TSB with organisms (growth control) and organism-free drug controls (sterility control) were used for comparison. Microtitre plates were incubated at 35 °C for 24 h, and MIC₅₀ and MIC₉₀ values were estimated by a colorimetric method using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino) carbonyl]-2H-tetrazolium hydroxide (XTT). Menadione [10 mM solution in acetone (99.9 % HPLC-grade)] was added to XTT (0.5 g l^–1) to give a 1 µM concentration of menadione and 100 µl of this was added to the wells of 96-well microtitre plates and incubated in the dark for 2 h. The plates were centrifuged for 5 min at 1932 g, 100 µl supernatant was transferred to a new microtitre plate and the colour was read at A₄₉₀ in a microtitre plate reader (Cerca et al., 2005). End points at 50 and 90 % inhibition of growth compared with the growth control were determined as MIC₅₀ and MIC₉₀ values, respectively. Experiments were carried out in duplicate on two different days and the means of the readings of XTT reduction were used to determine the MIC₅₀ and MIC₉₀.

MICs of TLF, AMB and FLC against C. albicans. MIC₅₀ and MIC₉₀ values were determined using the broth microdilution method as described by the CLSI (2002). C. albicans inoculum (100 µl) was added to 100 µl of serial twofold dilutions of the antifungal agent to be tested in 96-well microtitre plates. The final concentration of C. albicans was 0.25x10³–0.5x10³ c.f.u. ml^–1 in each well of the microtitre plate. Drug-free medium with organisms (growth control) and organism-free drug controls (sterility control) were used for comparison. Microtitre plates were incubated at 35 °C for 46 h, and MIC₅₀ and MIC₉₀ values were estimated by a colorimetric method using XTT as described above. Experiments were carried out in duplicate on two different days and the means of the readings of XTT reduction were used to determine MIC₅₀ and MIC₉₀ values.

Antimicrobial susceptibility testing of CoNS and C. albicans using antimicrobial combinations with TLF. Antimicrobial combinations of TLF with VAN or NAF were tested against CoNS, and combinations of TLF with AMB or FLC against C. albicans. An 8x8 chequerboard matrix of the drugs (50 µl each) in serial twofold dilutions including zero concentrations on the x- and the y-axes were set up in 96-well microtitre plates. As in the MIC determinations, 5 µl CoNS suspension (2.5x10⁴–7.5x10⁴ c.f.u. ml^–1) or 100 µl C. albicans suspension (0.5x10³–1x10³ c.f.u. ml^–1) was added to the wells containing the antimicrobial combinations. The plates were incubated at 35 °C for 24 h for CoNS and for 46 h for C. albicans. Inhibitory end points were determined by XTT reduction. Experiments were performed in duplicate on two different days and the means of readings of XTT reduction on each day were used to provide two data points for analyses.

Analyses of drug interactions in antimicrobial combinations. The inhibitory effects of the antimicrobials in combination with TLF were tabulated in constant ratios of each other including ratios of their MIC₅₀ values (equipotency ratios). The median effects principle was used to study drug interactions and estimate combination indices (CIs) and dose reduction indices (DRIs) (Chou, 2006).

The median effect equation is given by f_a/f_u=(D/D_m)^m, where f_a is the fraction affected by the drug, f_u is the fraction unaffected (f_u=1–f_a), D is the concentration of the drug, D_m is the median-effect dose (the dose of the drug that produces 50 % of the effect, ED₅₀) and m is the co-efficient signifying the shape of the dose–effect relationship, where m=1, m >1 and m <1 indicate hyperbolic, sigmoidal and flat-sigmoidal dose–effect curves, respectively.

The CI value is given by the equation CI=D₁/D_x1+D₂/D_x2, and the DRI for drug 1=D_x1/D₁ and for drug 2=D_x2/D₂, where D₁ and D₂ are doses of drug 1 and drug 2 in combination and D_x1 and D_x2 are doses of drug 1 and drug 2 that produce x % effect when used alone. A value of CI <1 indicates synergy, CI >1 indicates antagonism and CI=1 indicates an additive effect. The DRI determines by how many fold a drug dose can be reduced in a synergistic drug combination for a given inhibitory effect on the organism. Multiple drug-dose effect calculations were performed using CalcuSyn software (Biosoft) using the constant ratios of the drug combinations.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Antimicrobial susceptibility of clinical strains of CoNS and C. albicans

The MIC₅₀ values of TLF against CoNS ranged from 500 to 2000 µg ml^–1, but the MIC₉₀ values were beyond the range tested for all strains except CoNS 106F (Table 1). For C. albicans, the MIC₅₀ values of TLF ranged from 62.5 to 250 µg ml^–1, but the MIC₉₀ values were beyond the range tested for all three strains. The huge difference in concentrations between MIC₅₀ and MIC₉₀ values suggest a bacteriostatic effect rather than a bacteriocidal effect. Similar large differences were seen with the fungistatic FLC, where the MIC₉₀ values were 1000 times greater than the MIC₅₀ values. The differences between MIC₅₀ and MIC₉₀ for NAF (varying from the same concentration to 16-fold), vancomycin (same to 2-fold) and amphotericin (16–33-fold) were not as pronounced as that of TLF or FLC.

Antimicrobial combinations with TLF

There are many advantages of combining drugs in the treatment of infections, including enhancing efficacy, the ability to decrease the doses of antimicrobial agents (leading to a reduction in toxicity) and minimizing the development of resistance. The potential for the clinical use of lactoferrin in conjunction with antibiotics in the therapy or prevention of neonatal sepsis suggested that it would be useful to evaluate the drug interactions of TLF with antibiotics commonly used in neonatal practice.

Although many methods of evaluating synergy, an additive effect or antagonism in drug combinations exist, we used the median effects principle elucidated by Chou (2006), which is widely used in cancer and infection research (Berenbaum, 1989; Greco et al., 1995; Odds, 2003; Prichard et al., 1993). The advantage of this method is that it overcomes the assumption that drug interactions are linear across dosages and drug effects. There is no general equation that fits all of the dose–response curves and hence the need exists to evaluate results over a range of effects (50–90 % inhibition) (Martinez-Irujo et al., 1996). We evaluated drug combinations with TLF in a systematic manner at three different dose effects (ED₅₀, ED₇₅ and ED₉₀) and multiple drug-dose ratios that may be relevant clinically. We confirmed that our data fitted the median effects equation (mean r value 0.95), rendering calculation of the CIs and DRIs valid (Chou, 2006).

CIs for drug combinations with TLF. In the eight strains of CoNS tested, the TLF and NAF combination was synergistic (CI <1) over a wide range of drug-dose ratios at dose effects of 50, 75 and 90 % with very few exceptions (all exceptions were at ED₅₀: S. epidermidis ATCC 55133 at a ratio of 8000 : 1 and CoNS 103A at 64 000 : 1 and 32 000 : 1; Table 2). Similarly, the TLF and VAN combination was synergistic (CI <1) at multiple drug-dose ratios at ED₅₀, ED₇₅ and ED₉₀ with no exceptions.

The synergy of lactoferrin with antibiotics commonly used against isolates of organisms of clinical interest is of great clinical relevance, as it has the potential for more effective therapy against drug-resistant strains and for a reduction in drug dosage. Leitch & Willcox (1999b) reported that 1024 µg lactoferrin ml^–1 reduced the minimum bactericidal concentrations of VAN required to kill S. epidermidis twofold. Kuipers et al. (1999) reported significant co-operative activity of lactoferrin and apolactoferrin (iron-free) with FLC, AMB and 5-fluorocytosine against clinical Candida isolates. Peptides derived from human lactoferrin containing the first 11 N-terminal residues were effective in making fluconazole-resistant C. albicans sensitive to fluconazole (Leitch & Willcox, 1999a; Wakabayashi et al., 1996, 1998).

The mechanism of the synergistic effect of lactoferrin with antifungal and anti-staphylococcal agents has not been fully validated. It appears that the synergistic effect is not due to the iron-sequestering effects of lactoferrin, as both apolactoferrin and iron-saturated lactoferrin have similar activities in vitro (Kuipers et al., 1999). A direct effect on the cell membranes of Candida and staphylococci is more likely (Leitch & Willcox, 1999a; Xu et al., 1999). We speculate that the action of lactoferrin on cell membranes complements the action of other drugs and may be responsible for the synergy.

DRIs for drug combinations with TLF. For CoNS, the DRIs indicated a significant reduction in the doses of NAF and VAN required when used in combination with TLF for a given effect, but varied widely among the strains of CoNS tested and also among the different drug-dose ratios tested in a single strain (Table 4). The DRI ranges for NAF in combination with TLF were: 2.5–10.4 against S. epidermidis ATCC 55133; 5.3–14.3 against S. epidermidis 100H; 2.1–6.6 against S. epidermidis 101S; 11.7–176 against CoNS 102R; 1.7–7.3 against CoNS 103A; 1.7–4.3 against CoNS 104O; 3.7–90.3 against CoNS 105D; and 1.5–4.2 against CoNS 106F. The DRI ranges for VAN in combination with TLF were: 1.6–5.9 against S. epidermidis ATCC 55133; 2.2–6.4 against S. epidermidis 100H; 2.3–4.8 against S. epidermidis 101S; 2.7–9.2 against CoNS 102R; 1.2–3.2 against CoNS 103A; 1.6–4.1 against CoNS 104O; 1.8–4.2 against CoNS 105D; and 1.7–4.9 against CoNS 106F.

Limitations of the study

The limitations of this study follow the inherent variability of chequerboard assays and hence the need for repeated assays. We repeated our experiments in duplicate and on two different days, and the readings on any particular day were carried out under the same experimental conditions and were averaged, generating two sets of data for analysis. Chequerboard combinations used serial twofold dilutions of the drug, but the effects of the drug concentrations between the twofold dilutions were not evaluated, which may be valuable in future studies. We did not test concentrations higher than 8300 µg ml^–1 for TLF, although some studies using higher concentrations (Kuipers et al., 1999) have shown complete inhibition of Candida isolates. The general limitation of experiments in vitro in terms of correlation with effects in vivo that exist may be due to the physical environment as well as the participation of other host defence mechanisms. Antimicrobial combinations need to be evaluated in appropriate animal models using clinical isolates from organisms of interest.

Conclusions

The antimicrobial activity of lactoferrins in general, and TLF in particular, against common neonatal isolates of CoNS (including S. epidermidis) and C. albicans, and the synergistic effect with antibiotics commonly used in neonatal practice, make TLF a promising agent in the therapy and prevention of neonatal sepsis. Lactoferrin has been effective in animal models of infection and in polymicrobial infections in vivo. Clinical studies are needed to evaluate the potential efficacy of TLF in the treatment and prophylaxis of neonates against systemic infections to reduce mortality and morbidity.

	ACKNOWLEDGEMENTS

 
We acknowledge the help of Agennix for donating TLF for our research. We also thank C. Fernandes and Bhagvatula Moorthy for critically reviewing the manuscript.

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