J. Anim Sci. 2008. 86:66-71. doi:10.2527/jas.2007-0216
© 2008 American Society of Animal Science
Utilization of lactoferrin to fight antibiotic-resistant mammary gland pathogens1,2
P. Lacasse*,3,
K. Lauzon
,
M. S. Diarra
and
D. Petitclerc
* Agriculture and Agri-Food Canada, Dairy and Swine Research and Development Centre, PO Box 90 STN Lennoxville, Sherbrooke, Quebec, Canada J1M 1Z3;
and
Crea Biopharma Inc., Sherbrooke, Quebec, Canada; and
Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Agassiz, British Columbia, Canada
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Abstract
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The widespread use of antibiotics has lead to the increased presence of pathogens that are less susceptible to their antibacterial effect. Lactoferrin (Lf) is naturally produced by the mammary gland. Lactoferrin is the main whey protein in human milk and is also present in cow’s milk but at a much lower concentration than in human milk. This protein appears to have many biological functions, including antibacterial and antiinflammatory activities. The best-known effect of Lf is to bind iron that is essential for bacterial growth. However, the cationic nature of this protein also appears to be important for the antimicrobial activity of this protein. Lactoferrin has a weak antibacterial effect when used alone, but interestingly, Lf appears much more effective when used at low concentration in combination with several antibiotics. The most striking observation is that Lf increases the inhibitory activity of penicillin up to 4-fold in most penicillin-susceptible Staphylococcus aureus strains, whereas this increase was 4- to 16-fold in penicillin-resistant strains. Indeed, Lf reduces β-lactamase activity in S. aureus strains producing this enzyme. Transcription of β-lactamase gene is dramatically repressed in the presence of Lf. We evaluated the efficacy of intramammary treatments containing penicillin G or bovine Lf (bLf), or both, to cure chronic mastitis caused by a clinical isolate of S. aureus highly resistant to β-lactam antibiotics. In a first trial, mastitis was induced in lactating cows by injecting a low dose of S. aureus through the teat canal of all quarters. Bacterial cure rate was null for control quarters, 11.1% for bLf, 9.1% for penicillin, and 45.5% for the combination of bLf and penicillin. A second trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. Quarters were treated with 100,000 IU of penicillin G with or without 250 mg of bLf for 7 d. Bacterial cure rate was greater for the bLf + penicillin combination (33.3%) compared with penicillin alone (12.5%). In conclusion, bLf added to penicillin is an effective combination for the treatment of stable S. aureus infections resistant to β-lactam antibiotics.
Key Words: lactoferrin • mastitis • antimicrobial • β-lactamase
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INTRODUCTION
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The widespread use of antibiotics has led to the increased environmental presence of pathogens that are less susceptible to their antibacterial effect. This has resulted in infections that are more difficult to cure. The efficacy of conventional antibiotic treatments against pathogens such as Staphylococcus aureus is low (Wilson et al., 1999
). Penicillin and closely related antibiotics of the β-lactam family are the best weapons against staphylococci. However, the massive use of these antibiotics has led to a dramatic increase in pathogens that can produce an enzyme called β-lactamase that inactivates β-lactam antibiotics, thereby resulting in microbial resistance (Aarestrup and Jensen, 1998). Therefore, there is an urgent need to find new antimicrobials to treat bovine mastitis. In this context, antimicrobial proteins, such as lactoferrin (Lf), are of great interest.
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OVERVIEW
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Lactoferrin is found in milk, bile, saliva, tear, and polymorphonuclear cells granules (Schanbacher et al., 1993; Baker and Baker, 2005). Lactoferrin is the main whey protein in human milk (1 to 6 mg/mL), and it is also present in cow’s milk but at a much lower concentration (0.01 to 0.1 mg/mL) than in human milk. Nevertheless, Lf content of cow’s colostrum (1 to 2 mg/mL) and mammary involutive secretion (20 to 100 mg/mL; Nuijens et al., 1996) is much greater than in cow’s milk. In addition, the concentration of Lf in milk increases dramatically in milk after acute mastitis (Harmon et al., 1976).
Bovine Lf is an 80-kDa iron-binding glycoprotein of the transferrin family. Lactoferrin consists of a single polypeptide chain of 689 amino acids. The molecule is folded into 2 lobes, each of which is divided into 2 domains (Moore et al., 1997). In each lobe, an iron-binding site lies between the 2 domains. These binding sites have a very high affinity for Fe3+ ions with binding constants of human Lf (hLf) of about 1022 M (Baker et al., 2002). One feature that distinguishes Lf from transferrin is its ability to retain iron at low pH (Shimazaki et al., 1993). In addition to Fe3+, Lf can bind several other metals ions such as Ga3+, Al3+, Mn3+, Cu2+, and Zn2+ (Baker et al., 1994); however, the biological significance of this property is unknown. Interestingly, binding of iron induces a conformational change of Lf. Indeed, crystallographic studies have shown that, in absence of iron, the 2 domains enclosing the iron site can move apart, resulting in an open conformation. Conversely, binding of iron will lock the 2 domains together, giving a more compact closed conformation (Baker and Baker, 2005). Accordingly, conformational change of Lf has also been detected by phage display peptide libraries (Noppe et al., 2004).
Lactoferrin is positively charged with an isoelectric point of ~8 (Steijns and van Hooijdonk, 2000). This cationic nature must be a major factor in its ability to bind to anionic molecules, such as LPS, DNA, and RNA (Baker and Baker, 2005). A high concentration of positive charge is present in the N-terminal region of Lf. Pepsin hydrolysis of bovine Lf (bLf) can liberate a peptide (i.e., amino acid residues 17–41), termed lactoferricin (Lfcin), which contains this region and retains many activities of the intact protein (Gifford et al., 2005).
The bLf appears to have many biological functions, including bacteriostatic, bactericidal, antiinflammatory, and immunomodulatory activities. The best-known effect of Lf is to bind iron that is essential for bacterial growth. This results in low iron availability and therefore inhibits the growth of bacteria. Originally, the antimicrobial activity of Lf was attributed entirely to its iron-sequestering capabilities. However, Lfcin, which cannot bind iron, has a more potent bactericidal effect than Lf itself. The presence of positive charge appears important for antimicrobial activity of bLf because reduction of the charge by acetylation reduces the antibacterial activity, whereas an increase of the charge by amidation increases the antibacterial activity (Pan et al., 2007). Nuijens et al. (1996) proposed that bacteriostasis is the result of iron sequestration, whereas the bactericidal activity is related to the binding of Lf to bacterial components (e.g., lipopolysaccharide, porins, DNA) that disrupt bacterial function and integrity.
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ANTIBACTERIAL ACTIVITY OF LACTOFERRIN AGAINST MASTITIS PATHOGENS
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Several in vitro studies have investigated antibacterial activity of Lf against strains of bacteria causing bovine mastitis. Growth of mastitis causing coliform bacteria is slowed down in media supplemented with more than 1 mg/mL of iron-deprived bLf (Bishop et al., 1976; Nonnecke and Smith, 1984b; Kutila et al., 2003). Accordingly, growth inhibition of these bacteria in whey collected during the dry period was proportional to the Lf concentration observed in whey (Nonnecke and Smith, 1984a). However, the growth inhibition can be reversed by addition of ferric iron or sodium citrate (Bishop et al., 1976; Nonnecke and Smith, 1984a,b) suggesting that iron restriction was the main mechanism of bLf action. Growth of staphylococci was inhibited to a lower extent by bLf (Nonnecke and Smith 1984b; Kutila et al., 2003), which can be related to the fact that S. aureus is able to grow in the presence of an extremely low (0.04 µM) iron concentration (Trivier and Courcol, 1996) or in the presence of a high concentration of ethylenediamine di-O-hydroxyphenylacetic acid, a powerful iron chelator (Diarra et al., 2002b). According to these results, bLf probably contributes to the resistance to intramammary infection during the dry period, but alone, its therapeutic potential appears low.
We have explored the possibility of combining bLf or Lfcin with classical antibiotics (Diarra et al., 2002a,c) against bovine bacterial pathogens. Minimal inhibitory concentrations (MIC) of Lf, Lfcin, penicillin, and combinations of Lf or Lfcin with penicillin were determined for 15 S. aureus strains. Lactoferrin alone demonstrated weak inhibitory activity against these strains with MIC greater than 25 mg/mL. The MIC of Lfcin was 256 µg/mL for all strains. The fractional inhibitory concentration index indicated a synergistic effect between Lf and penicillin. Combination of Lf with penicillin increased the inhibitory activity of penicillin by 2- to 4-fold and reduced the growth rate in S. aureus strains tested, whereas the increase in the inhibitory activity of Lf by penicillin was 16- to 64-fold. Addition of iron to the medium containing a combination of penicillin and Lf had no effect on bacterial growth inhibition. Electron microscopy revealed that concentration below the MIC of penicillin induced important ultrastructural alterations in penicillin susceptible strains, which were further enhanced by the presence of Lf. In the penicillin-resistant S. aureus strain SHY97-4320, 8 µg/mL of penicillin G alone had no visible effect, but when combined with 1 mg/mL of Lf, morphological changes were similar to those induced by penicillin in a susceptible strain (Diarra et al., 2003b). The therapeutic potential of bLf or bLfcin in combination with cefazolin and neomycin against Escherichia coli and Klebsiella pneumoniae strains isolated from clinical bovine mastitis cases was also evaluated in our laboratory (Diarra et al., 2002c). Alone, bLf did not affect the growth of E. coli but, at concentration 
0.5 mg/mL, it partially inhibited the growth of K. pneumoniae. In E. coli, no synergistic inhibitory effect was observed between cefazolin and Lf, but complete growth inhibition was obtained when 0.5 mg/mL of Lf was combined to 1/2 MIC neomycin. Accordingly, bLfcin acted synergistically with subMIC of cefazolin and neomycin to reduce the growth of E. coli and with cefazolin to inhibit growth of K. pneumoniae. In agreement with our results, Sanchez and Watts (1999) reported a synergistic effect between bLf and novobiocin against E. coli. These authors have attributed this effect to the binding of Lf to LPS causing a greater permeability of the membrane allowing the antibiotic to penetrate the bacteria. These results indicate that bLf or bLfcin can increase the antibacterial activity of traditional antibiotics against gram-negative and gram-positive mastitis bacterial pathogens.
We further investigated the interaction between penicillin and Lf or Lfcin by evaluating the effect of Lf or Lfcin on β-lactamase production in S. aureus resistant to penicillin G (Diarra et al., 2000, 2001). The quantitative spectrophotometric method using nitrocefin showed that Lf and Lfcin reduced β-lactamase activity in S. aureus strains producing this enzyme (Figure 1). Transcription of β-lactamase gene was dramatically repressed in the presence of Lf or Lfcin in the media as determined by real-time reverse transcription-PCR [D. Petitclerc, M. S. Diarra, D. Labrecque (Dairy and Swine R&D Centre, Sherbrooke, QC, Canada), and P. Lacasse, unpublished results]. These results indicate that Lf or Lfcin can affect β-lactamase gene expression and can be used in combination with β-lactams to increase the antibacterial activity of these antibiotics against resistant S. aureus strains.
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Figure 1. Beta-lactamase activity, measured as a change in optical density at 486 nm, in Staphylococcus aureus strain SHY97-4320 after 30 or 60 min preincubation with 1 mg/mL of bovine lactoferrin and exposed to 8 µg/mL of penicillin G (PG) during 4 h of incubation at 37° C. Values are means of 3 separate experiments. Data are unpublished data of D. Petitclerc, M. S. Diarra, D. Labrecque (Dairy and Swine R&D Centre, Sherbrooke, QC, Canada), and P. Lacasse.
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TREATMENT OF BOVINE MASTITIS WITH LACTOFERRIN
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Kutila et al. (2004) have compared the efficacy of bLf and enrofloxacin for the treatment of E. coli mastitis. They observed a trend for a more rapid elimination in enrofloxacin-treated quarters than bLf-treated quarters. However, in absence of untreated quarters, the contribution of the bLf treatment to the clearance of the bacteria is difficult to determine. Kai et al. (2002a) have treated quarters infected by S. aureus during the dry period with bLf or the antibiotic cefazolin. Both treatments caused a rapid reduction of the bacterial population, but 14 d after injection, the number of bacteria was lower in bLf-treated quarters. However, those results were obtained from a limited number of animals and need to be confirmed in larger studies.
The efficacy of intramammary treatments containing penicillin G and bLf, alone or in combination, was evaluated using a model of experimentally induced chronic bovine mastitis caused by a clinical isolate of S. aureus that is highly resistant to β-lactam antibiotics (Petitclerc et al., 2007). In the first trial, chronic mastitis was induced in 19 late-lactating cows by injecting a low dose of S. aureus through the teat canal of all quarters. After 15 d, cows with stable infections in their 4 quarters had their mammary quarters randomly assigned, within cow, to 1 of 4 treatments: 1) citrate buffer; 2) 100,000 IU of penicillin G; 3) 1 g of bLf, and 4) 1 g of bLf + 100,000 IU of penicillin G. Treatments were repeated twice daily for 5 d. The number of bacteria shed by control quarters remained stable for the whole period. During the treatment period, all quarters that received bLf, penicillin, or both had a significant decrease in milk bacterial count (Figure 2). As early as 2 d after the last treatment (d 7), the number of milk cfu in quarters treated with penicillin or bLf alone were no longer different from their pretreatments levels, whereas milk cfu remained reduced in quarters that received the combined treatment. This residual effect was linked to the clearing out of infection in several of the quarters of the combined treatment. Indeed, cure rate was null for control quarters, 11.1% for bLf, 9.1% for penicillin, and 45.5% for the bLf + penicillin combination.
A second trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. One month prior to dry-off, twenty 7-mo-pregnant cows, regrouped by dates of calving, were infected in all 4 quarters. Once infections were established, cows were dried off abruptly. After calving, aseptic milk samples were collected separately from all quarters for 4 wk to monitor infection. Mammary quarters from enrolled cows were then randomly assigned, within cow, to treatment with 100,000 IU of penicillin G or 250 mg of bLf + 100,000 IU of penicillin G. Treatments were administered intramammarily twice daily for 7 d. In agreement with the first experiment, bLf enhanced the antimicrobial activity of penicillin. Cure rate was greater for the bLf + penicillin G combination (33.3%) compared with penicillin G alone (12.5%).
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EFFECT OF LACTOFERRIN ON HOST DEFENSE
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Ideally, antimicrobials should combine good antibacterial activity and the capacity to work in association with the host defense system. Many effects of Lf on immune functions have been reported and sometimes appear contradictory. Indeed, immunostimulatory proinflammatory and antiinflammatory activities have been reported. Therefore, extrapolation from other animal models is difficult.
Kai et al. (2002b) have reported that bLf increases phagocytosis by bovine phagocytes in the presence of fresh serum but not heat-inactivated serum. We have investigated the effects of bLf alone or in combination with penicillin G on the phagocytic activity of bovine polymorphonuclear leukocytes against S. aureus (Diarra et al., 2003b). Susceptibility of S. aureus to phagocytosis was decreased in the presence of penicillin in the medium. In a kinetic study, Lf alone did not affect phagocytosis, but when used with penicillin, Lf reversed the negative effect of this antibiotic on phagocytosis. In addition, the killing ability of polymorphonuclear leukocytes was enhanced by Lf in the presence or absence of penicillin. Using an epithelial invasion assay, Lf alone or in combination with penicillin reduced the invasion of mammary epithelial cells in culture by S. aureus (Diarra et al., 2003b). These results indicate that bovine Lf, alone or in combination with penicillin G, enhances S. aureus susceptibility to immunodefense mechanisms, which can be beneficial in the treatment of S. aureus infections.
Intramammary administration of bLf caused an important increase in somatic cell count (SCC; Kai et al., 2002a; Petitclerc et al., 2007). In the latter study, 1 g of bLf contained only 0.2 µg of endotoxin and such a low amount of endotoxin is unlikely to cause such a large rise in SCC. We have compared the administration of a commercial bLf, with or without an additional purification on healthy cows, and have observed large SCC increases for both Lf preparations (Figure 3). These observations support the view that bLf may be active in modulation and regulation of macrophages, lymphocytes, and neutrophil function (Sordillo et al., 1997), thus attracting more neutrophils into the mammary gland. The significance of this effect for the clearance of infection has not been established.
The activation of leukocytes by LPS and the massive production of free radicals contribute to the pathogenesis of coliform mastitis. Baveye et al. (2000) reported that hLf inhibits the binding of LPS to L-selectin and the subsequent production of reactive oxygen species by human neutrophils. Accordingly, protective effects of Lf have been reported on gut mucosal integrity (Kruzel et al., 2000) and endotoxin lethal shock (Lee et al., 1998) during LPS-induced endotoxemia. Guillen et al. (2000) have also postulated that Lf binding of iron prevent free radical formation at site of inflammation. Nevertheless, using an in vitro model of neutrophil-induced mammary epithelial cell damage (Boulanger et al., 2002), we have never observed any protective effect of Lf [K. Lauzon, X. Zhao (McGill University, Montreal, QC, Canada), and P. Lacasse, unpublished results].
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CONCLUSION
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Lactoferrin is a protein that does far more than just bind iron. In vitro and in vivo results indicate that Lf is active against mammary pathogens, but its intrinsic activity is probably too low to constitute a viable treatment by itself. However, intramammary therapy using a combination of bLf with antibiotics could enhance significantly the bacteriological cure rate compared with antibiotic treatment alone. Therefore, bLf added to an antibiotic might be an effective combination for the treatment of chronic infections caused by bacterial pathogens resistant to antibiotics. The effects of Lf on the bovine immune system may also contribute to the treatment of mastitis and should be more closely investigated.
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Footnotes
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1 Presented at the Eighth International Workshop on the Biology of Lactation in Farm Animals held in Pirassununga, Brazil, August 21–23, 2006. 
2 Dairy and Swine Research and Development Centre Contribution No. 925.
3 Corresponding author: lacassep{at}agr.gc.ca
Received for publication April 16, 2007.
Accepted for publication June 2, 2007.
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G. E. Dahl
The Eighth International Workshop on the Biology of Lactation in Farm Animals: Introduction
J Anim Sci,
March 1, 2008;
86(13_suppl):
1 - 2.
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Abstract
INTRODUCTION
), bLf (
), PG (
), or bLf + PG (x) for 5 consecutive days. For the horizontal axis, the time of the first intramammary infusion of treatments was d 0. Data are presented as values transformed to log10 (± SEM). Figure taken from Petitclerc et al. (2007); with permission.
), commercial bLf (
). For the horizontal axis, the time of the first intramammary infusion of treatments was d 0. Data are presented as means. From unpublished data of P. Lacasse, K. Lauzon, and D. Petitclerc.