Subinhibitory concentrations of florfenicol enhance the adherence of florfenicol-susceptible and florfenicol-resistant Staphylococcus aureus

Maren Blickwede1, Peter Valentin-Weigand2 and Stefan Schwarz1,*

1 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystrasse 10, 31535 Neustadt-Mariensee; 2 Institut für Mikrobiologie, Zentrum für Infektionsmedizin, Tierärztliche Hochschule Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany

Keywords: invasion , fibronectin , surface hydrophobicity

Sir,

The ability of Staphylococcus aureus to adhere to surfaces of host tissues is thought to be essential for colonization and establishment of infections.1 There is increasing evidence that subinhibitory concentrations of antibiotics interfere with microbial adherence to host cells. In this study, the effect of subinhibitory concentrations of the fluorinated chloramphenicol derivative florfenicol on adherence properties of florfenicol-susceptible and florfenicol-resistant S. aureus was investigated.

The florfenicol-susceptible strain S. aureus Newman (ATCC 25904) and its florfenicol-resistant derivative carrying the cfr-erm(33)-spc multiresistance plasmid pSCFS1 from Staphylococcus sciuri2 were cultivated for 20 h at 37°C in brain heart infusion broth (BHI, Oxoid, Wesel, Germany) and in BHI supplemented with either 0.2 mg/L florfenicol (Essex Tierarznei, Munich, Germany) or 2 mg/L florfenicol (=1/20 and 1/2 MIC for S. aureus Newman, respectively). In addition, S. aureus Newman:pSCFS1 was cultivated in BHI supplemented with 64 mg/L florfenicol (=1/2 MIC for S. aureus Newman:pSCFS1). MICs of florfenicol were determined by broth microdilution according to the NCCLS guideline M31-A2.3 Adherence and invasion assays using 5 x 106 bacteria per experiment and confluent monolayers of HEp-2 cells (~3 x 105 cells per well) were carried out as described by Dziewanowska et al.4 The results were recorded as percentage of cfu of adherent or intracellular bacteria. For microscopic confirmation, adherence assays were carried out on culture slides containing ~2 x 105 HEp-2 cells. Washed monolayers with adherent bacteria were fixed with 0.37% formaldehyde (Sigma, Taufkirchen, Germany) at 4°C, washed with PBS and covered with blocking buffer [10% fetal calf serum (Sigma) in PBS] for 1 h. Adherent staphylococci were detected by use of a rabbit-anti-staphylococcal antibody, an FITC-conjugated goat-anti-rabbit IgG antibody (Dianova, Hamburg, Germany) and subsequent immunofluorescence microscopy. Bacteria-per-epithelial cell ratios were determined by counting manually the number of bacteria adherent to epithelial cells in three microscopic fields with ~50 epithelial cells per field.

Growth in the presence of low subinhibitory concentrations of florfenicol, such as 0.2 mg/L for S. aureus Newman as well as 0.2 and 2 mg/L for S. aureus Newman:pSCFS1, did not change significantly the adherence patterns of both strains compared with the untreated controls. However, when grown in the presence of the strain-specific 1/2 MIC, both strains adhered significantly better (P≤0.05) to HEp-2 cells (Table 1). Although invasion also increased significantly when the bacteria were treated with the strain-specific 1/2 MIC, the percentages of invading bacteria ranged between 0.1% and 0.28% (Table 1) and thus were negligible in view of the percentages of adherent bacteria. This observation was in good accordance with that of Dziewanowska et al.4 who showed that S. aureus strain Newman is less invasive than other S. aureus strains which exhibit comparable adherence to epithelial cells. Thus, virtually all bacteria detected after release from the epithelial cells could be considered as truly adherent bacteria. Quantification of the adherent bacteria per epithelial cell by immunochemical microscopic evaluation yielded results that correlated well with those obtained by the plating technique. A four-fold increase in the number of adherent bacteria per epithelial cell was detected in case of the florfenicol-susceptible S. aureus Newman whereas an eight-fold increase was seen in the case of the florfenicol-resistant S. aureus Newman:pSCFS1 (Table 1).


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Table 1. Effect of subinhibitory concentrations of florfenicol on adherence and invasion properties of florfenicol-susceptible S. aureus strain Newman and florfenicol-resistant S. aureus strain Newman:pSCFS1

 
Bacterial adhesion can be the result of either hydrophobic interactions between the bacteria and the host cells, binding of bacteria to specific ligands, e.g. fibronectin, or a combination of both. Therefore, florfenicol-treated staphylococci and untreated controls were investigated for their surface hydrophobicity by a hydrocarbon adherence assay5 and for their ability to bind to fibronectin-coated micotitre plates (1 µg of fibronectin per well).4 Independently of the florfenicol concentration used, both strains showed a non-significant decrease in their surface hydrophobicity (Table 1). In contrast, the fibronectin-binding profiles of both strains grown in the presence of the strain-specific 1/2 MIC of florfenicol exhibited significant four- to five-fold increases (Table 1). Based on this finding, we assume that binding to fibronectin plays a relevant role in the increased adherence of the staphylococci to HEp-2 cells, rather than non-specific hydrophobic interactions.

The observations made in this study differ from those of a previous study on tetracycline-treated S. aureus in which increased adherence was directly correlated with significantly increased hydrophobicity.6 However, the results of our study also showed parallels to a study in which pretreatment of S. aureus with sub-MIC levels of ciprofloxacin gave an induced expression of fibronectin binding proteins and also resulted in increased adhesion of the staphylococci.7 Although further work is needed to elucidate the mechanisms leading to the observed changes in the adherence patterns, our data demonstrate that florfenicol concentrations in the range of strain-specific 1/2 MIC enhance the adherence of florfenicol-susceptible and also florfenicol-resistant S. aureus to epithelial cells, and thus may alter virulence properties of this pathogen.

Acknowledgements

We wish to thank Laurentiu Benga for his expert introduction into the adherence experiments and Gunter Amtsberg for the staphylococcal antibody. This work was supported by the Deutsche Forschungsgemeinschaft (GK 745).

Footnotes

* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; Email: stefan.schwarz{at}fal.de

References

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3 . National Committee for Clinical Laboratory Standards (2002). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals—Second Edition: Approved Standard M31-A2. NCCLS, Wayne, PA, USA.

4 . Dziewanowska, K., Patti, J. M., Deobald, C. F. et al. (1999). Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells. Infection and Immunity 67, 4673–8.[Abstract/Free Full Text]

5 . Rosenberg, M., Gutnick, D. & Rosenberg, E. (1980). Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiology Letters 9, 29–33.[CrossRef][ISI]

6 . Schwarz, S. (1994). Increased cell wall thickness and its effects on potential virulence properties of tetracycline-resistant Staphylococcus aureus. Medical Microbiology Letters 3, 16–22.[ISI]

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