1 Department of Medical Microbiology, University Hospital of North Norway, Tromsø, Norway; 2 Department of Microbiology and Virology, University of Tromsø, Tromsø, Norway; 3 Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany; 4 Department of Medicine and Medical Microbiology/Immunology, University of Wisconsin Medical School, Madison, Wisconsin, USA; 5 Northern Norway Regional Health Authority, Bodø, Norway
Received 4 August 2005; returned 15 September 2005; revised 28 September 2005; accepted 28 September 2005
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Abstract |
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Methods: A broth microdilution technique was used for susceptibility testing to determine the MIC of lactoferricin B for SCVs with three different auxotrophisms (haemin, menadione or thymidine) and their isogenic parent strains. Both clinical isolates and genetically defined mutants were used. The internalization of lactoferricin B in a hemB mutant and the respective parent strain was studied using transmission electron microscopy and immunogold labelling.
Results: All SCVs showed reduced susceptibility to lactoferricin B irrespective of their auxotrophy compared with their isogenic parent strains. The MIC for all SCVs was >256 mg/L, whereas the MICs for the parent strains ranged from 16256 mg/L. Surprisingly, the hemB mutant contained significantly more lactoferricin B intracellularly than the respective parent strain.
Conclusions: The resistance mechanism of SCVs towards the antimicrobial peptide lactoferricin B is presumably caused by the metabolic changes present in SCVs rather than by a changed transmembrane potential of SCVs or reduced uptake of the peptide.
Keywords: cellular uptake , electron microscopy , immunolabelling , transmembrane potential , metabolic resistance , antimicrobial resistance , resistance mechanism
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Introduction |
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Antimicrobial peptides or host defence peptides are an integral part of the host defence against invading microorganisms. Since SCVs are able to survive and persist in vivo, resistance to these peptides might be a prerequisite. However, SCVs are not universally resistant to all antimicrobial peptides. Magainin and human neutrophil defensin-1 have similar activity against SCVs and normal S. aureus, whereas SCVs are resistant to protamine and thrombin-induced platelet microbicidal protein.4,5 Sadowska et al.5 have shown that under exposure to subMIC concentrations of the antimicrobial peptide protamine, both haemin- and menadione-dependent SCVs emerge.
Lactoferricin B is a multifunctional cationic antimicrobial peptide derived from bovine lactoferrin, with antibacterial, antiviral, antiparasitic, antitumour and immunomodulating activities. The antibacterial mode of action of lactoferricin B is likely to be multitargeted involving the outer surface, cytoplasmic membrane and intracellular targets. Resistance to lactoferricin B has been possible to induce in S. aureus,9 and proteases have previously been described to be involved in susceptibility to lactoferricin B.10 So far, the SCV phenotype was not observed in the lactoferricin B-resistant S. aureus. However, since SCVs are frequently unstable and media were not appropriate to culture SCVs, they might have been missed without actively searching for SCVs.9
To determine whether SCVs are resistant to lactoferricin B, the susceptibility of SCVs with different auxotrophies was determined. Furthermore, the internalization of lactoferricin B was compared between a hemB mutant mimicking the SCV phenotype11 and the isogenic parent strain using transmission electron microscopy and immunogold labelling.
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Materials and methods |
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Bacterial strains used in this study are listed in Table 1. MuellerHinton (MH) broth (Difco, Detroit, USA) was used as growth and assay medium for all strains. Erythromycin (5 mg/L) (Astra, Sødertalje, Sweden) was used in agar plates and overnight broth cultures for the genetically defined mutants (hemB::ermB; menD::ermC) to maintain pure cultures of the mutants. The menD mutant was constructed as described in Bates et al.6 except that the COL strain was used as the parent strain.
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The MIC was determined using a standard broth microdilution technique. Briefly, 2-fold dilutions (2256 mg/L) of lactoferricin B in double distilled water (125 µL) were added to microtitre plates (Nunc, Roskilde, Denmark) and exponentially growing bacteria (125 µL) were added resulting in a final bacterial concentration of 106 cfu/mL. The MIC was determined as the lowest concentration that inhibited growth after 24 and 48 h for the normal strains and after 48 h of incubation for the SCVs. Lactoferricin B was prepared by pepsin digestion of bovine lactoferrin and purified by reverse phase HPLC (Centre for Food Technology, Queensland, Australia).
Immunolabelling, transmission electron microscopy and determination of intracellular content of lactoferricin B
The intracellular localization and amount of lactoferricin B in S. aureus 8325-4 and the isogenic hemB mutant I-10 exposed to 30 mg/L lactoferricin B for 30 min were determined as previously described.12 The MannWhitney nonparametric test was used for comparison of intracellular peptide content.
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Results and discussion |
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It has previously been shown that the transmembrane potential of the hemB mutant I-10 dropped immediately (to values below 100 mV) when glucose expired and other nutritions such as acetate and lactate did not allow further growth.7 To examine if an intact electron transport chain and transmembrane potential is involved in internalization of lactoferricin B, the hemB mutant I-10 and the parent strain 8325-4 were exposed to 30 mg/L lactoferricin B for 30 min. Electron microscopy and immunogold labelling showed a significantly higher amount of intracellular labelling of lactoferricin B in the hemB mutant than in the parent strain (P < 0.0001) (Figure 1). This suggests that lactoferricin B does not require an intact transmembrane potential to interact with and cross the cytoplasmic membrane. Thus, the resistance mechanism in SCVs is not due to reduced uptake of the peptide as observed for aminoglycosides for haemin- and menadione-auxotroph SCVs.7
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These findings show that SCVs (haemin-, menadione- or thymidine-auxotrophs) are resistant to the antimicrobial peptide lactoferricin B, and that the resistance mechanism may be a form of a metabolic resistance and not due to reduced uptake. These results also suggest that lactoferricin B crosses the membrane through passive diffusion or promotes the uptake itself. Further studies are warranted to determine the exact mechanism of resistance.
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Acknowledgements |
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References |
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