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Antimicrobial Agents and Chemotherapy, May 2003, p. 1739-1741, Vol. 47, No. 5
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.5.1739-1741.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Burkholderia Is Highly Resistant to Human Beta-Defensin 3

Hany Sahly,1* Sabine Schubert,1 Jürgen Harder,2 Peter Rautenberg,1 Uwe Ullmann,1 Jens Schröder,2 and Rainer Podschun1

Department of Medical Microbiology and Virology,1 Clinical Research Unit, Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany2

Received 31 October 2002/ Returned for modification 9 December 2002/ Accepted 29 January 2003


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ABSTRACT
 
The bactericidal activity of the novel beta-defensin hBD-3 against 28 species and 55 strains of gram-positive cocci and gram-negative fermentative and nonfermentative rods was tested. All strains proved to be highly or intermediately susceptible to hBD-3 (minimal bactericidal concentration [MBC], <=50 µg/ml), except for Burkholderia cepacia, for all 23 tested strains of which MBCs were >100 µg/ml.


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TEXT
 
Human beta-defensins play important roles in innate immune responses in mucosal surfaces and skin, probably by virtue of their antimicrobial activity (1, 3, 6, 16, 18, 22, 24, 25). Beta-defensins 1 and 2 have been shown to have potent antibacterial activity against gram-negative but not against gram-positive bacteria (9, 24, 26). In contrast, the novel human beta-defensin-3 (hBD-3) was recently found to be potent against gram-positive and gram-negative bacteria, including Burkholderia cepacia, which resists both beta-defensins 1 and 2. However, only two studies have expressly evaluated the antimicrobial potential of hBD-3, and each of these considered only a restricted number of bacterial isolates (seven and eight, respectively) (4, 10).

In the present study the susceptibility to hBD-3 of a comprehensive and representative panel of reference and clinical isolates of gram-positive cocci as well as gram-negative fermentative and nonfermentative rods comprising 28 species and a total of 55 strains was tested based on the minimal bactericidal concentration (MBC) and the lethal dose for >=50% of bacteria (LD50). The strains were either reference strains obtained from the American Type Culture Collection or the National Collection of Type Cultures (London, United Kingdom) or clinical strains isolated from human tracheal secretions or from blood culture (Table 1). Additionally, isolates belonging to a B. cepacia complex strain panel that has been described as appropriate for diagnostic and experimental purposes with regard to this genus (17) were obtained from the Belgium Coordinated Collections of Microorganisms/Laboratorium Microbiologie (BCCM/LMG) (Table 2). hBD-3 was synthesized either recombinantly, by cloning cDNA encoding the 45 amino acids containing the natural form of hBD-3 into the pET-30c expression vector (Novagen), which contains an N-terminal His tag sequence allowing purification of the fusion protein with a nickel affinity column, or chemically (Jerini Bio Tools GmbH, Berlin, Germany) as described previously (10). Test isolates were grown for 2 to 3 h in brain heart infusion broth at 36 ± 1°C, washed three times in 10 mM sodium phosphate buffer (pH 7.4), and adjusted to 104 to 105 bacteria/ml. A 100-µl volume of the bacterial suspension was mixed with 10 µl of hBD-3 solution (range of final concentrations tested, 0.0125 to 100 µg/ml) and incubated at 36 ± 1°C. After 2 h, CFU were determined. Bacterial suspensions supplemented with 10 µl of phosphate buffer or with 10 µl of 0.01% acetic acid instead of hBD-3 served as negative controls. Results are given either as MBC (>=99.9% killing) or as LD50s. Arbitrarily, a strain was defined as sensitive to hBD-3 if MBC levels were <10 µg/ml, as intermediately sensitive if MBC levels were 10 to 100 µg/ml, or as resistant at MBC levels of >100 µg/ml. hBD-3 showed broad activity against all bacterial groups tested. All tested strains of gram-positive cocci, gram-negative fermentative rods, and gram-negative nonfermentative rods were shown to be highly susceptible ((MBC and LD50, <=6.25 µg/ml) except Serratia marcescens and Aeromonas hydrophila, both of which exhibited intermediate sensitivity (MBC, 50 µg/ml) (Table 1). In contrast to the previous findings of other groups (4, 10), the genus Burkholderia alone was resistant to hBD-3; for all 23 Burkholderia strains tested, MBCs and LD50s were >100 µg/ml, except for 3 isolates for which LD50s were <=12.5 µg/ml (Table 2).


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  		TABLE 1. MBCs and LD50s of hBD-3 against a reference panel of clinically relevant bacterial species


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  		TABLE 2. MBCs and LD50s of hBD-3 against the Burkholderia complexa

Our findings suggest that hBD-3 has the potential to play an important role in the clearance of opportunistic gram-negative and gram-positive bacteria. The broad range of microbicidal activity shown by hBD-3 is very promising with regard to its utility as a therapeutic alternative to antibiotic chemotherapeutics, particularly because resistance to antimicrobial peptides in general cannot be induced under physiological conditions (8). The applicability of hBD-3, however, is restricted by the high resistance of the B. cepacia complex.

The resistance of the B. cepacia complex to hBD-3 might be in part the cause for the relatively high isolation rates of B. cepacia from immunocompromised patients with severe underlying diseases such as cystic fibrosis (5, 11).

The results of the present study support the hypotheses that (i) unlike defensins 1 and 2, whose microbicidal activity is directed predominantly against gram-negative bacteria (9, 15, 24), hBD-3 has broad activity against both gram-positive and gram-negative bacteria, including nonfermentative rods other than Burkholderia and (ii) the genus Burkholderia possesses an intrinsic resistance to hBD-3. These results contradict those of Garcia et al. (4) showing B. cepacia to be rather susceptible to hBD-3 (MIC, 6.6 µg/ml). This discrepancy in the results of two working groups may be due in part to differences in the methodologies applied. While Garcia et al. measured bacterial growth by means of optical density, we quantitatively determined viable counts as a gauge of microbicidal efficacy. Furthermore, the former group measured the ability of hBD-3 to inhibit growth of the bacteria (MIC), while our study considered the bactericidal capacity (MBC). Additionally, Garcia et al. based their conclusion on results achieved with only two B. cepacia strains, while we studied 23 reference strains, all of which were found to be resistant to hBD-3.

Burkholderia spp. are known to resist cationic peptides, as shown for polymyxin B (19), cathelicidin peptides (23), and human beta-defensins 1 and 2 (2). It has been suggested that the resistance of gram-negative bacteria to cationic peptides is in part due to structural peculiarities of the lipopolysaccharide (LPS) anchored in the bacterial outer membrane (7, 20). As to polymyxin B, several studies have shown that the various gram-negative bacteria with a priori or acquired resistance to this antibiotic peptide exhibit extensive cationic substitution of LPS by 2-amino-4-deoxy-L-arabino-pentopyranose (L-Arap4N) (12, 13, 21). This substitution produces a reduction in the net negative charge of LPS, thus impairing the affinity of the bacterial surface for positively charged cationic peptides. Interestingly, Isshiki et al. have shown that LPS of B. cepacia is constitutively substituted by ß-L-Arap4N-(1->8)-{alpha}-Kop(2->4)-KDO (14).

The question of whether the substitution of LPS by L-Arap4N, which reduces the negative net charge of the outer membrane, does indeed constitute the molecular basis for the resistance of B. cepacia to both polymyxin B and hBD-3 needs further clarification. This could be achieved by generating a B. cepacia L-Arap4N-deficient mutant and then testing its susceptibility to hBD-3.


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ACKNOWLEDGMENTS
 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 617.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medical Microbiology and Virology, University Hospital Schleswig-Holstein, Campus Kiel, Brunswiker Str. 4, 24105 Kiel, Germany. Phone: 49 431 597 3316. Fax: 49 431 597 3296. E-mail: sahly{at}medmicrobio.uni-kiel.de. Back


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Antimicrobial Agents and Chemotherapy, May 2003, p. 1739-1741, Vol. 47, No. 5
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.5.1739-1741.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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