Alcoholic ingredients in skin disinfectants increase biofilm expression of Staphylococcus epidermidis

Johannes K.-M. Knobloch,*, Matthias A. Horstkotte, Holger Rohde, Paul-Michael Kaulfers and Dietrich Mack

Institut für Medizinische Mikrobiologie und Immunologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The pathogenesis of Staphylococcus epidermidis is correlated with biofilm formation. We investigated the effect of three common alcoholic skin disinfectants, ethanol, n-propanol and isopropanol, on the biofilm formation of 37 clinical, icaADBC-positive S. epidermidis isolates. In alcohol-supplemented media 18 strains displayed increased biofilm expression. Sixteen of 19 strains were generally incapable of biofilm formation. In three representative isolates, the increase in biofilm formation was paralleled by increased polysaccharide intercellular adhesin synthesis. Regarding the widespread use of alcoholic skin disinfectants, it is possible that the alcohol-inducible biofilm phenotype of S. epidermidis could add to the development of foreign body-related infections.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In recent years Staphylococcus epidermidis has been isolated with increasing frequency as a causative pathogen of nosocomial infections, which are often foreign body related. The major pathogenic factor is the ability to form biofilms on polymeric surfaces.1 Essential for cell accumulation is the expression of a polysaccharide intercellular adhesin (PIA), which mediates cell-to-cell adhesion and is synthesized by the icaADBC gene products.1 Recently, it was shown that expression of icaADBC is dependent on RsbU, a positive regulator of {alpha}B, and that ethanol stress induces biofilm formation.2,3

Alcoholic skin disinfectants are frequently used, resulting in a high extent of bacterial elimination; however, small numbers of bacteria still survive on skin.4 We therefore investigated the effect of the three most common alcoholic ingredients of skin disinfectants: ethanol, n-propanol and isopropanol, on biofilm formation of S. epidermidis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial strains, growth conditions and phenotypic characterization

S. epidermidis strains 1457 and 8400 were used as reference organisms.5 One hundred and thirty-eight clinical isolates of S. epidermidis were sampled during a 12 month period from February 1998 to January 1999. The isolates were from blood cultures (n = 56), central venous or peritoneal dialysis catheters (74) and other relevant clinical specimens (eight). Isolates were identified using standard microbiological techniques.5 Biofilm production was measured by a semi-quantitative adherence assay in trypticase soy broth (TSBBBL; Becton Dickinson, Cockeysville, MD, USA).3,6 Biofilm formation was classified into strongly biofilm positive (OD570 >= 1), low grade biofilm-positive (0.1 <= OD570 < 1) and biofilm negative (OD570 < 0.1). For analysis of the influence of disinfectants, TSBBBL was supplemented with different concentrations of ethanol [1, 2, 4 or 6% (v/v)], n-propanol [0.5, 1, 2 or 4% (v/v)], isopropanol [1, 2, 4 or 6% (v/v)], benzalkonium chloride (0.1, 0.01 or 0.001 µg/mL) and chlorhexidine (0.1, 0.01 or 0.001 µg/mL), respectively. Increased biofilm formation due to the different alcohols was defined as at least OD570 0.2 when the strain was primary biofilm negative, or at least doubling of the OD570 for low-grade biofilm-positive strains.

Quantification of PIA synthesis

Bacterial extracts and cell supernatants were prepared as described previously.3 PIA concentrations were determined by a co-agglutination assay with PIA-specific antiserum.6 Immunochemical variant PIA (PIAv), expressed by strain CNS161, was detected using an antiserum raised against S. epidermidis 7415, which expressed a polysaccharide not reactive with anti-PIA antiserum, absorbed with the isogenic biofilm-negative Tn917 mutant 1457-M11.5

Genotypic characterization

Chromosomal DNA of S. epidermidis was prepared and amplification of DNA fragments was performed as described previously.3,6 Amplification of a part of icaB was performed as described previously.6 Oligonucleotides specific for icaR (icaR for 5'-ACTGGTAAGTCCGTCAAGT-3') and icaC (icaC-H rev 5'-CAAGCACATACATAAGCCATAG-3') of S. epidermidis (GenBank accession no. U43366) were synthesized by MWG Biotech (Munich, Germany).7

Pulsed-field gel electrophoresis (PFGE) was performed as described previously for analysis of clonal relationships.2 Strains with identical PFGE patterns were only included for further analysis when they were isolated from different patients and not temporally related.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
RsbU, a positive regulator of the alternative sigma factor {alpha}B, positively controls biofilm formation in S. epidermidis.3 Thereby, ethanol stress leads to increased PIA production and biofilm formation in the reference strains S. epidermidis 1457 and 8400.3 To further investigate this phenotype, 138 clinical isolates of S. epidermidis were analysed for the presence of icaADBC by icaB-specific PCR, and the ability for biofilm formation in a standard biofilm assay. Sixty-eight strains (49%) were icaB positive; 70 strains (51%) displayed no icaB signal. Of the icaB-positive strains, 29 (43%) were strongly biofilm positive, 17 (25%) displayed low-grade biofilm expression and 22 (32%) were biofilm negative. Owing to limitations of photometers, an increase of biofilm formation in strongly biofilm-producing S. epidermidis strains could barely be detected.3 We therefore further investigated 39 biofilm-negative and low-grade biofilm-producing strains. PFGE analysis of these strains revealed 33 different patterns (Table 1Go). PFGE patterns 2 and 11, and patterns 6 and 15, were represented by two and three strains, respectively. Two of the pattern 6 strains were excluded from further analysis because their isolation was temporally related, indicating nosocomial transmission.


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Table 1. Phenotypic and genotypic properties of S. epidermidis strains investigated
 
Biofilm formation was analysed in TSBBBL supplemented with different concentrations of ethanol, n-propanol and isopropanol. In 18 of 37 strains, increased biofilm formation was induced by ethanol, whereas 15 and 14 of these strains were inducible by n-propanol and isopropanol, respectively (Table 2Go). In the group containing low-grade biofilm-producing strains, 14 of 17 displayed increased biofilm formation, whereas only three strains were not inducible. The induction patterns of three representative clinical isolates are shown in the Figure (a–cGo). For these strains, expression of PIA was investigated using the optimum-inducing alcohol concentrations. For CNS27 and CNS156, increased cell-associated and supernatant PIA concentrations were detected (FigureGo). CNS161 did not react with the anti-PIA antiserum. Increased expression of PIAv was detected with absorbed antiserum raised against S. epidermidis 7415 (FigureGo). Using this antiserum, similar antigen concentrations were detected with strains CNS27 and CNS156, indicating that PIA and PIAv structures are closely related (FigureGo).


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Table 2. Induction of biofilm formation in 37 icaADBC-positive, biofilm-negative or low-grade biofilm-positive clinical isolates by different alcohols
 


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Figure. Induction of biofilm formation (a–c) in three representative icaADBC-positive clinical isolates in TSBBBL supplemented with ethanol [EtOH (a); {blacksquare}, TSB; , 1%; , 2%; , 4%; , 6%], n-propanol [n-prop.; (b); {blacksquare}, TSB; , 0.5%; , 1%; , 2%; , 4%] and isopropanol [isoprop.; (c); {blacksquare}, TSB; , 1%; , 2%; , 4%; , 6%]. The primary biofilm-negative strain 1 (CNS27) displayed strong biofilm induction by all three alcohols investigated (a–c). The primary biofilm-positive strain 2 (CNS156) is also strongly inducible by all tested alcohols even at high alcohol concentrations (a–c), whereas strain 3 (CNS161) displayed weaker induction only for ethanol and n-propanol (a, b). Additional induction of strain 3 was only detectable for lower alcohol concentrations in the media (a, b). Results of representative experiments are shown. Error bars indicate standard errors. PIA expression [(d–f); {blacksquare}, biofilm; , PIA cells; , PIAv cells; , PIA supernatant; , PIAv supernatant] by S. epidermidis after induction of biofilm formation by ethanol, n-propanol and isopropanol. S. epidermidis strains CNS27 (d), CNS156 (e) and CNS161 (f) were grown in TSBBBL or in TSBBBL supplemented with the respective alcohol concentration as stated. Bacterial extracts and culture supernatants were prepared, and PIA and PIAv concentrations were determined by co-agglutination using specific antisera.

 
Sixteen of 20 biofilm-negative strains were not inducible by alcohol supplementation, indicating a general incapability of biofilm formation. To exclude functional genetic defects, icaADBC of these strains was amplified spanning icaR to icaC. No difference in size of the fragments was observed, indicating an intact icaADBC locus (data not shown). However, the possibility of point mutations cannot be excluded.

In media supplemented with increasing alcohol concentrations, the growth rate of the cells was decreased, whereas biofilm formation was increased for inducible S. epidermidis strains (data not shown). n-Propanol displayed the strongest effect, inhibiting cell growth totally at a concentration of 4%. In media supplemented with 6% ethanol or 6% isopropanol, some strains still displayed slow growth and a few strains showed increased biofilm formation (FigureGo).

Strains displaying increased biofilm formation were investigated for the influence of chlorhexidine and benzalkonium chloride on biofilm expression. For both compounds no positive influence on biofilm formation was observed at subinhibitory concentrations, while biofilm formation decreased in parallel with decrease in the growth rate (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
S. epidermidis biofilm formation and PIA synthesis is quantitatively modulated by environmental factors.1,3 The increasing biofilm formation due to ethanol supplementation by S. epidermidis reference strains suggests that this phenomenon could be of relevance for the development of nosocomial S. epidermidis infections in the clinical setting, where alcoholic skin disinfectants are routinely used. In the present study we characterized the effect of three alcohols, ethanol, n-propanol and isopropanol, on biofilm formation by 37 unrelated clinical isolates of S. epidermidis, which harbour the icaADBC gene locus and displayed no or low biofilm formation.3

A total 18 of 37 (49%) strains were inducible to increased biofilm formation by at least one of the alcohols. Increased biofilm formation was linked to increased production of PIA in strains CNS27 and CNS156. Interestingly, CNS161 turned out to be a strain expressing PIAv, an apparent immunochemical variant of PIA. Absorbed antisera raised against the PIAv-producing S. epidermidis 7415 was cross-reactive to PIA, indicating that both antigens are structurally related. PIAv expression was also stimulated by the different alcohols, indicating that expression of PIAv is regulated in a similar manner to PIA.3

In the group of primary low-grade biofilm-forming S. epidermidis strains, 82% displayed an inducible phenotype due to at least one of the alcohols tested. Surprisingly, only 20% of the primary biofilm-negative icaADBC- positive S. epidermidis displayed an increase in biofilm formation after alcohol supplementation, indicating a general incapability of these strains to form biofilm. No difference in size could be detected in PCR fragments spanning icaRADBC of all non-inducible strains, indicating an intact icaADBC locus. Apparently, insertions by insertion sequences in icaADBC8,9 occur infrequently.

In addition to icaADBC there are at least three independent gene loci influencing PIA expression and biofilm formation.2 These gene loci could also be a target for mutation or deletion leading to a biofilm-negative phenotype. A rsbU-insertion mutant was biofilm-negative but could be induced by ethanol stimulation to produce biofilm and PIA.3 Similar mutations could lead to the alcohol-dependent biofilm formation of biofilm-negative, icaADBC- positive clinical isolates.

The results with reference strains and primary biofilm-positive strains support the conclusion that a comparable percentage of the strongly biofilm-producing isolates display biofilm induction by alcohols. Therefore, >60% of the icaADBC-positive clinical isolates are predicted to be inducible in biofilm formation by alcohols in the environment.

For cutaneous antisepsis before the insertion of catheters and before surgery, as well as for surgical hand disinfection, alcoholic disinfectants are recommended.4,10 In clinical use the tested alcohols are bactericidal. However, small numbers of bacteria remain viable after skin disinfection.4 In addition, recolonization of the skin occurs, owing to the skin flora close to the disinfected area. Almost nothing is known about the diffusion and the following evaporation of the alcohols in the deeper layers of the skin. Therefore, the time course of an alcohol concentration gradient may reach conditions comparable to our test conditions for a relevant time period. It is reasonable to speculate that such conditions could confer a positive selective pressure on biofilm-positive S. epidermidis in deep skin layers and skin around the disinfection site, promoting the occurrence of medical device-related infections.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Rainer Laufs for his continuous support. This work was supported by a grant of the Deutsche Forschungsgemeinschaft (to D.M.).


    Notes
 
* Corresponding author. Tel: +49-40-42803-3147; Fax: +49-40-42803-4881; E-mail: knobloch{at}uke.uni-hamburg.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Mack, D., Bartscht, K., Dobinsky, S., Horstkotte, M. A., Kiel, K., Knobloch, J. K. M. et al. (2000). Staphylococcal factors involved in adhesion and biofilm formation on biomaterials. In Handbook for Studying Bacterial Adhesion: Principles, Methods, and Applications, 1st edn, (An, Y. H. & Friedman, R. J., Eds), pp. 307–30. Humana Press, Totowa, NJ.

2 . Mack, D., Rohde, H., Dobinsky, S., Riedewald, J., Nedelmann, M., Knobloch, J. K. M. et al. (2000). Identification of three essential regulatory gene loci governing expression of the Staphylococcus epidermidis polysaccharide intercellular adhesin and biofilm formation. Infection and Immunity 68, 3799–807.[Abstract/Free Full Text]

3 . Knobloch, J. K. M., Bartscht, K., Sabottke, A., Rohde, H., Feucht, H. H. & Mack, D. (2001). Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress. Journal of Bacteriology 183, 2624–33.[Abstract/Free Full Text]

4 . Rotter, M. L. (1996). Hand washing and hand disinfection. In Hospital Epidemiology and Infection Control, 1st edn, (Mayhall, C. G., Ed.), pp. 1052–68. Williams and Wilkins, Baltimore, MD.

5 . Mack, D., Haeder, M., Siemssen, N. & Laufs, R. (1996). Association of biofilm production of coagulase-negative staphylococci with expression of a specific polysaccharide intercellular adhesin. Journal of Infectious Diseases 174, 881–4.[ISI][Medline]

6 . Mack, D., Bartscht, K., Fischer, C., Rohde, H., de Grahl, C., Dobinsky, S. et al. (2001). Genetic and biochemical analysis of Staphylococcus epidermidis biofilm accumulation. Methods in Enzymology 336, 215–39.[ISI][Medline]

7 . Gerke, C., Kraft, A., Süssmuth, R., Schweitzer, O. & Götz, F. (1998). Characterization of the N-acetylglucosaminyltransferase activity involved in the biosynthesis of the Staphylococcus epidermidis polysaccharide intercellular adhesin. Journal of Biological Chemistry 273, 18586–93.[Abstract/Free Full Text]

8 . Ziebuhr, W., Krimmer, V., Rachid, S., Lößner, I., Götz, F. & Hacker, J. (1999). A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Molecular Microbiology 32, 345–56.[ISI][Medline]

9 . Rohde, H., Knobloch, J. K. M., Horstkotte, M. A. & Mack, D. (2001). Correlation of biofilm expression types of Staphylococcus epidermidis with polysaccharide intercellular adhesin synthesis: evidence for involvement of icaADBC genotype-independent factors. Medical Microbiology and Immunology (Berlin) 190, 105–12.[ISI][Medline]

10 . Pearson, M. L. (1996). Guideline for prevention of intravascular device-related infections. Hospital Infection Control Practices Advisory Committee. Infection Control and Hospital Epidemiology 17, 438–73.[ISI][Medline]

Received 29 May 2001; returned 14 September 2001; revised 13 December 2001; accepted 21 December 2001