JAC Advance Access originally published online on January 25, 2006
Journal of Antimicrobial Chemotherapy 2006 57(3):562-565; doi:10.1093/jac/dkl003
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Activity of human ß-defensins 2 and 3 against ESBL-producing Klebsiella strains
1 Institute of Infection Medicine, Faculty of Medicine, University of Kiel, Brunswiker Strasse 4, 24105 Kiel, Germany; 2 Clinical Research Unit, Department of Dermatology, Medical School, University of Kiel, Schittenhelmstrasse 7, 24105 Kiel, Germany; 3 PLANTON GmbH, am Kiel-Kanal 44, 24106 Kiel, Germany; 4 National Centre for Antimicrobials and Infection Control, Statens Serum Institut, Artillerivej 5, Copenhagen, Denmark
* Corresponding author. Tel: +49-431-597-3316; Fax: +49-431-597-3296; E-mail: sahly{at}infmed.uni-kiel.de
Received 4 August 2005; returned 27 October 2005; revised 6 December 2005; accepted 23 December 2005
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Abstract |
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Objectives: To test the bactericidal activity of human ß-defensins (hBDs) 2 and 3 against extended-spectrum ß-lactamase (ESBL)-producing Klebsiella strains.
Methods: Thirty-six Klebsiella pneumoniae and seventeen Klebsiella oxytoca ESBL-producing isolates from nosocomial infections were tested. The bactericidal activity of recombinantly synthesized hBD-2 and -3 was tested and the results were given either as lethal doses killing 
90% of bacteria (LD90s) or as MBCs (99.9% killing).
Results: Except for one intermediately susceptible strain (MBC = 25 mg/L), all other ESBL-producing strains were highly susceptible to both defensins (LD90s and MBCs 
12.5mg/L).
Conclusions: The results underline the high efficacy of hBD-2 and -3 against ESBL-producing Klebsiella, making both defensins attractive candidates as antimicrobial agents to combat these increasingly troublesome bacteria.
Keywords: multidrug resistance , innate immunity , Klebsiella spp
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Introduction |
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Klebsiella was once known primarily as a pathogen that caused severe pyogenic, community-acquired pneumonia and bacteraemia mainly affecting chronic alcoholics and having a high fatality rate if untreated.1,2 Today, however, the vast majority of Klebsiella infections are nosocomial. As an opportunistic pathogen, Klebsiella primarily attacks immunocompromised individuals who are hospitalized and have severe underlying diseases.1 Klebsiella infections are observed in almost any body site, although infections of the urinary and respiratory tracts predominate. Depending on the type of infection and study, their prevalence ranges from 3 to 17% of all such infections, placing them among the eight most important pathogens in hospitals and second only to Escherichia coli as the most common cause of Gram-negative sepsis.3
Especially threatening is the dramatic worldwide spread of Klebsiella strains whose ability to produce plasmid-coded extended-spectrum ß-lactamases (ESBLs) renders them resistant to the bactericidal activity of third-generation cephalosporins. Various types of ESBL, especially TEM and SHV enzymes, have been described worldwide.4 Recent surveys show that ESBL-producing Klebsiella pneumoniae isolates account for 
8% of ESBL-producing strains in the USA, 23% in Europe, 25% in the Western Pacific and 45% in Latin America.5 At present the first-line therapy against ESBL-producing strains is carbapenems. The emergence of carbapenem-resistant strains, however, has increasingly limited the use of these antibiotics for the treatment of ESBL-caused infections6 and prompted an intensive search for new approaches for prevention and treatment of such bacteria.
Defensins are a unique family of cysteine-rich cationic peptides with three or four disulphide bridges. They are isolated from mammals, insects and plants.7 Because most of them are expressed consecutively, exhibit broad-spectrum antimicrobial activity and are produced by cell tissues (e.g. skin and mucosal tissues) commonly exposed to a wide variety of microorganisms, they are thought to constitute one of the major arms of innate immunity.7
Human ß-defensins (hBDs) 2 and 3 are recently discovered cationic antimicrobial peptides that play an important role in the innate immune response of skin and mucosal surfaces, mainly by eliciting direct bactericidal activity. Whereas the bactericidal activity of hBD-2 is restricted to Gram-negative fermentative and non-fermentative bacteria,8 we have recently shown that hBD-3 has a broad antimicrobial potential against fermentative and non-fermentative Gram-negative bacilli including E. coli, K. pneumoniae, Serratia marcescens, Citrobacter freundii, Aeromonas hydrophila, Pseudomonas and Acinetobacter spp. and Stenotrophomonas maltophilia as well as against Gram-positive cocci including Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes.9 Because both defensins are highly salt sensitive and are affected by the supplementation with nutrient ingredients, conventional determination of the MICs of both defensins is not possible. Thus, MBCs or lethal doses killing 90% of bacteria (LD90s) in 10 mM phosphate buffer without salt are used to gauge antibacterial activity (H. Sahly, S. Schubert, R. Podschun, unpublished data).
In the present study we investigated the antibacterial potential of hBD-2 and -3 against ESBL-producing Klebsiella strains.
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Materials and methods |
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Defensins
hBD-2 and -3 were synthesized recombinantly by cloning cDNA encoding the amino acids of the natural form of the defensins into the expression vector pET-30c (Novagen/Merck Biosciences, Nottingham, UK), which contains an N-terminal His-Tag sequence allowing purification of the fusion protein with a nickel-affinity column and cleavage with subsequent HPLC separation of the mature ß-defensin as described previously.9
Bacterial strains and typing procedures
A total of 36 K. pneumoniae and 17 Klebsiella oxytoca isolates were tested. All strains were isolated between 1994 and 2000 from relevant specimens of patients hospitalized at different hospitals in six European countries (Denmark, England, France, Germany, Spain and Turkey) with infections meeting the Center for Disease Control criteria for nosocomial pneumonia, urinary tract infection, primary bloodstream infection or soft tissue infections. The origin of each strain was noted and only one strain per patient was included. The strains were identified by means of the API 20E system (bioMérieux, Nürtingen, Germany) and additional macro tube tests. ESBLs were detected according to recommendations of the CSLI (formerly NCCLS).10 All strains were initially screened for ESBL production by cefpodoxime, ceftazidime, cefotaxime or ceftriaxone antimicrobial discs and confirmed phenotypically by testing for synergy between ceftazidime, cefotaxime or ceftriaxone and clavulanic acid. To analyse clonality, all 53 strains were subjected to pulsed-field gel electrophoresis (PFGE) and K-serotyped as described previously.11 The PFGE patterns were categorized into four different categories (indistinguishable, closely related, possibly related and different) as described by Tenover et al.12 Strains that were indistinguishable, closely related or possibly related according to Tenover's criteria for the analysis of PFGE pattern and identical K-serotype were regarded as clonal.
To determine the ESBL types in each clonal and non-clonal strain, primers specific for blaTEM and blaSHV were applied. The PCR products were sequenced and the sequences processed in BioNumerics GeneBuilder (Applied Maths BVBA, Belgium). The consensus sequence was compared with the Entrez Nucleotides database using NCBI Blast as described previously.11
Antibacterial activity testing
Susceptibility of the strains to the defensins was defined as the MBC (99.9% killing) as described previously.9 Briefly, after a 2–3 h growth period in brain heart infusion broth at 36 ± 1°C, the strains were washed three times in 10 mM sodium phosphate buffer (pH 7.4) and adjusted to 104–105 bacteria per mL in the same buffer, because the antimicrobial activity of both defensins significantly decreased in the presence of broth or additional salts other than those available in the 10 mM sodium phosphate buffer (data not shown). Ten microlitres of defensin solution (range of final concentrations tested: 0.0125–100 mg/L) was added to 100 µL of the bacterial suspension and incubated at 36 ± 1°C for 2 h before colony forming units were determined. Bacterial suspensions supplemented with 10 µL of phosphate buffer or with 10 µL of 0.01% acetic acid instead of the defensins served as negative controls. The antibacterial activities of the defensins were given either as LD90s or MBCs (99.9% killing). A strain was arbitrarily defined as susceptible to both defensins if it had MBCs 12.5 mg/L, as intermediately susceptible if it had MBCs of 25–100 mg/L or as resistant if it had MBCs > 100 mg/L.
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Results and discussion |
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The results of the clonality analysis and susceptibility testing are summarized in Table 1. In total, five clones were defined and the number of strains in each clone ranged from two to eight strains. All clonal strains included in this study showed either an identical or closely related PFGE pattern. Among these strains 28 capsular types and 12 different ESBL types were identified. In six strains ESBL production was confirmed but the ESBL types were non-typeable (Table 1).
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Except for the CF 1 strain, which exhibited an MBC of 25 mg/L, all other strains were highly susceptible to both defensins; their MBCs did not exceed 12.5 mg/L. The MBC of hBD-2 ranged from 1.56 to 12.5 mg/L, and the MBC of hBD-3 ranged from 0.39 to 12.5 mg/L.
These results suggest that hBD-2 and -3 possess high antibacterial potential and harmonize with previous data showing both defensins to be highly effective against Gram-negative bacteria.8,9 Significantly, the proven antibacterial activity against ESBL-producing multidrug-resistant strains makes them attractive candidates for application as novel antibiotics. This notion is of particular interest as ESBL strains have emerged that are also resistant to carbapenems, the only remaining therapeutic alternative against ESBL producers.
In preliminary studies of single strains we could show that hBD-3 has efficient antibacterial activity against other multidrug-resistant Gram-positive and Gram-negative bacteria such as Pseudomonas aeruginosa, vancomycin-resistant Enterococcus faecium and methicillin-resistant S. aureus, whereas hBD-2 is efficient at killing P. aeruginosa isolates and ESBL-producing E. coli (H. Sahly, S. Schubert, J. Harder, M. Kleine, J. M. Schröder, R. Podschun, unpublished data). Thus, the antibacterial potential of hBD-2 and -3 could also be effective against other Gram-positive and Gram-negative multidrug-resistant bacteria. However, these results need to be confirmed in systematic studies of a large and representative number of isolates of each of these multiresistant groups. Moreover, the sensitivity of these cationic antibacterial peptides to various salt concentrations and preparation conditions might pose the biggest challenge to producing therapeutically effective drugs meeting pharmacokinetic standards for systemic application. For both defensins, laboratory-scale recombinant production has been established, whereas large-scale production to satisfy commercial needs is presently being established for hBD-2.
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No declarations were made by the authors of this paper.
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Acknowledgements |
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This study was supported by the ‘Deutsche Forschungsgemeinschaft (SFB 617)’.
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References |
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1. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998; 11: 589–603.
2. Ko WC, Paterson DL, Sagnimeni AJ et al. Community-acquired Klebsiella pneumoniae bacteremia: global differences in clinical patterns. Emerg Infect Dis 2002; 8: 160–6.[ISI][Medline]
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4. Gniadkowski M. Evolution and epidemiology of extended-spectrum ß-lactamases (ESBLs) and ESBL-producing microorganisms. Clin Microbiol Infect 2001; 7: 597–608.[CrossRef][ISI][Medline]
5. Winokur PL, Canton R, Casellas JM et al. Variations in the prevalence of strains expressing an extended-spectrum ß-lactamase phenotype and characterization of isolates from Europe, the Americas, and the Western Pacific region. Clin Infect Dis 2001; 32: 94–103.
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Cao VT, Arlet G, Ericsson BM et al. Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration. J Antimicrob Chemother 2000; 46: 895–900.
7. Crovella S, Antcheva N, Zelezetsky I et al. Primate ß-defensins—structure, function and evolution. Curr Protein Pept Sci 2005; 6: 7–21.[Medline]
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Sahly H, Schubert S, Harder J et al. Human ß-defensin 3 has a potent antimicrobial effect against Gram-negative and Gram-positive bacteria, except the Burkholderia cepacia complex. Antimicrob Agents Chemother 2003; 47: 1739–41.
10. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing. Supplemental Tables M100-S12. NCCLS, Wayne, PA, USA, 2002.
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Sahly H, Aucken H, Benedi VJ et al. Increased serum resistance in Klebsiella pneumoniae strains producing extended-spectrum ß-lactamases. Antimicrob Agents Chemother 2004; 48: 3477–82.
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