Post-antibiotic effect of the antimicrobial peptide lactoferricin on Escherichia coli and Staphylococcus aureus

Hanne H. Hauklanda,* and Lars H. Vorlanda,b

a Department of Medical Microbiology, University Hospital of Tromsø, N-9038 Tromsø; b Department of Medical Microbiology, Institute of Medical Biology, University of Tromsø, Tromsø, Norway


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Lactoferricin is an antimicrobial peptide generated by gastric pepsin cleavage of lactoferrin. A possible post-antibiotic effect (PAE) of bovine lactoferricin (Lfcin B) and two shorter peptide derivatives against Staphylococcus aureus and Escherichia coli was studied. A drug removal technique involving centrifugation and washing was used. No PAE was found for Lfcin B against these two bacteria. The shorter derivatives had a short PAE against E. coli. They had a short negative PAE against S. aureus. In conclusion, the overall PAE is not overwhelming, but the small differences found between the different peptide–bacteria combinations could indicate that different peptide mechanisms of action might be present.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Lactoferricin is a potent antimicrobial peptide released from the N-terminal part of lactoferrin by gastric pepsin cleavage. Lactoferricin B (Lfcin B) consists of 25 amino acids corresponding to residues 17–41 from the N-terminus of bovine lactoferrin.1 The primary sequence of Lfcin B contains many hydrophobic and positively charged residues, indicating that it may interact with biological membranes.2 It is thought that its main activity is on the cytoplasmic membrane.1,3 We report in this study a possible post-antibiotic effect (PAE) of Lfcin B (17–41) and two shorter derivatives, L-Lfcin B (17–31) and d-Lfcin B (17–31) on Escherichia coli and Staphylococcus aureus.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Strains

We used the bacterial strains E. coli ATCC 25922 and S. aureus ATCC 25923, and two clinical isolates each of E. coli and of S. aureus. The bacteria were stored at –70°C. The bacteria were grown in 2% bacto peptone water (Difco, Detroit, MI, USA) pH 6.8. We used bacteria in the mid-logarithmic growth phase.

Peptides

Synthetic Lfcin B (17–41) was a gift from Wayne Bellamy (Nutritional Science Laboratory, Morinaga Milk Industry Co. Ltd, Kanawaga 228, Japan). The sequence of this peptide is NH2-Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe-COOH. Two derivatives, l-Lfcin B (17–31) and d-Lfcin B (17–31), consisting of the same 15 amino acids, all l or all d amino acids, respectively, were synthesized on solid phase using fluorenylmethoxycarbonyl (Fmoc) chemistry, analysed and purified by high-performance liquid chromatography. The sequence of these peptides is NH2-Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-COOH. The peptides were diluted in sterile, distilled water to a concentration 100 times higher than the desired test concentrations and stored at –20°C. The final concentrations used were 5 x MIC for

each combination of peptide and bacteria.

MIC and MBC determination

Determination of MIC and MBC was carried out in parallel as described by Vorland et al.4

PAE procedure

The drug removal technique described by McDonald et al.5 involving centrifugation and washing was used, with some adjustments. The test procedures were performed in a total volume of 1 mL. The bacteria were grown at 37°C with shaking in a water bath to log phase (107–108 cfu/mL). Two tubes (A, the test tube; B, the control tube) were treated the same throughout the procedure. The concentration of bacteria in each test tube was 107 cfu/mL at time zero. The tubes were incubated at 37°C with shaking in a water bath for 1 h. At the end of this exposure period the tubes were centrifuged for 10 min at 1200g. The bacterial suspensions were washed and centrifuged three times. Following this drug removal procedure, the bacterial suspension was transferred to polystyrene round-bottomed tubes. To check whether the bacterial growth in the control tube might be inhibited by the density of bacteria, in some cases we established a second control tube (b) after the drug removal procedure. This tube contained 100 µL of the bacterial solution from control tube B, diluted 1/10 in 900 µL pre-warmed growth medium. The tubes were further incubated under the same conditions until turbidity was observed by eye.

The bacteria were counted at time zero, before and after the washing procedure and every 1 h thereafter. At each sampling time 10 µL of bacterial solution was transferred to cold 0.9% NaCl, serially diluted and plated on PDM agar (Paper Disk Method Antibiotic Sensitivity Medium II; AB Biodisk, Solna, Sweden). The plates were incubated over-night at 37°C. The colonies were counted and the cfu/mL and the logarithms of the cfu/mL calculated. The PAE was estimated using the formula PAE = T – C, where T is the time required for the test culture to increase 1 log10 (10-fold) from the count observed immediately after the drug removal and C is the time required for 1 log10 increase in the control tube. Each experiment, with its control, was carried out in triplicate.

Competition assay

Competition assays between teichoic acid (TA) and the short peptides were performed as described by Vorland et al.4 We used the bacterial strain S. aureus ATCC 25923 at 1 x 104 cfu/mL. The concentration range for TA in the microtitre tray was 0–1 mg/L. The concentration range for the peptides was 0–40 mg/L.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The results of the PAE experiments are shown in Tables 1 and 2GoGo. Lfcin B (17–41) showed no significant PAE on S. aureus or E. coli. The shorter peptide derivatives had a short PAE on E. coli. We found a slightly negative PAE on S. aureus for the short derivatives Lfcin B (17–31), more pronounced for the peptide made of all l amino acids than its counterparts made of all d amino acids.


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Table 1. Mean PAE of various lactoferricins against S. aureus strains ATCC 25923 and clinical isolates 63481/99 and 63135/99
 

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Table 2. Mean PAE of various lactoferricins against E. coli strains ATCC 25922 and clinical isolates 9784/99 and 9785/99
 
In the competition assays we found that with increasing amount of TA, an increasing amount of both the d- and the l-Lfcin B (17–31) was necessary to obtain a growth-inhibiting effect on the bacteria.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Factors that may affect PAE are described by Craig et al.6 It is suggested that the degree of PAE might be related to the degree of cellular damage done by the antibiotic to the bacterial cell. The more damaged the cell is, the longer the time needed for it to repair. We found no PAE for Lfcin B (17–41) on E. coli or S. aureus. This might indicate that the damage to the cells is not very profound, or at least very easily repaired. We have shown previously, however, that Lfcin B (17–41) kills the cells rather rapidly and at concentrations lower than those used in this setting.7 Cociancich et al.8 have suggested that perhaps all cells are not hit by the peptides. This might be owing to an aggregation of the peptides, rendering the active part of them much smaller. The cells that are hit could be effectively killed, but cells that are not hit may divide, unaffected by the peptides.

The shorter peptide derivatives had a short negative PAE on S. aureus, the l form more than the d form. Lfcin B (17–41) binds to teichoic acid.4 It is suggested that autolysin binds to TA.9 Our competition studies indicated that the d- and the l-Lfcin B (17–31) bind to TA. The binding of these peptides to TA may inhibit the binding of autolysin to TA. Septa may be made, but the cells will not separate owing to the missing action of autolysins. This may result in the formation of pseudomulticellular bacteria, which may contain many genomes, as described by Lorian.10 As the peptide is removed, autolysin may bind to TA, thereby completing the cell separation.

Proteases may break down the peptides, thereby releasing amino acids as an extra substrate source in this culture, giving the bacteria an advantage as compared with the control culture, thereby creating a negative PAE. A peptide consisting of all l amino acids is more easily broken down by proteases than a peptide made of all d amino acids. Our finding of the more negative PAE for the l form is consistent with this presumption.

The negative PAE might be a false result owing to a bacterial density that is potentially too high in the control tube, giving these bacteria suboptimal growth conditions. Dilution of these control cultures (tubes b), however, gave the same results, contradicting this assumption.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Manuela Krämer and Kjersti Sandvik for skilful technical assistance, and Hilde Ulvatne for assistance in preparing the manuscript. This study was supported by grants from ALPHARMA A/S, Oslo, Norway and The Norwegian Research Council, Oslo, Norway.


    Notes
 
* Corresponding author. Tel: +47-77-62-70-29; Fax: +47-77-62-70-15; E-mail: mlabhh{at}rito.no Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., Kawase, K. & Tomita, M. (1992). Identification of the bactericidal domain of lactoferrin. Biochimica et Biophysica Acta 1121, 130–6.[ISI][Medline]

2 . Hwang, P. M., Zhou, N., Shan, X., Arrowsmith, C. H. & Vogel, H. J. (1998). Three-dimensional solution structure of lactoferricin B, an antimicrobial peptide derived from bovine lactoferrin. Biochemistry 37, 4288–98.[ISI][Medline]

3 . Ulvatne, H., Haukland, H. H., Olsvik, O. & Vorland, L. H. (2001). Lactoferricin B causes depolarization of the cytoplasmic membrane of Escherichia coli ATCC 25922 and fusion of negatively charged liposomes. FEBS Letters 492, 62–5.[ISI][Medline]

4 . Vorland, L. H., Ulvatne, H., Rekdal, O. & Svendsen, J. S. (1999). Initial binding sites of antimicrobial peptides in Staphylococcus aureus and Escherichia coli. Scandinavian Journal of Infectious Diseases 31, 467–73.[ISI][Medline]

5 . McDonald, P. J., Craig, W. A. & Kunin, C. M. (1977). Persistent effect of antibiotics on Staphylococcus aureus after exposure for limited periods of time. Journal of Infectious Diseases 135, 217–23.[ISI][Medline]

6 . Craig, W. A., Gudmundsson, S. & Lorian, V. (1996). Postantibiotic effect. In Antibiotics in Laboratory Medicine, 4th edn, (Lorian, V., Ed.), pp. 296–329. Williams & Wilkins, Baltimore, MD.

7 . Ulvatne, H. & Vorland, L. H. (2001). The bactericidal kinetics of three lactoferricins against Escherichia coli and Staphylococcus aureus. Scandinavian Journal of Infectious Diseases 33, 507–11.[ISI][Medline]

8 . Cociancich, S., Ghazi, A., Hetru, C., Hoffmann, J. A. & Letellier, L. (1993). Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. Journal of Biological Chemistry 268, 19239–45.[Abstract/Free Full Text]

9 . Giudicelli, S. & Tomasz, A. (1984). Attachment of pneumococcal autolysin to wall teichoic acids, an essential step in enzymatic wall degradation. Journal of Bacteriology 158, 1188–90.[ISI][Medline]

10 . Lorian, V. (1975). Some effect of subinhibitory concentrations of penicillin on the structure and division of staphylococci. Antimicrobial Agents and Chemotherapy 7, 864–7.[ISI][Medline]

Received 11 December 2000; returned 18 March 2001; revised 14 May 2001; accepted 5 July 2001





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