The effect of human lactoferrin on the MICs of doxycycline and rifampicin for Burkholderia cepacia and Pseudomonas aeruginosa strains

M. Alkawash, M. Head, I. Alshami and J. S. Soothill*

Department of Pathological Sciences, University of Manchester, 2nd Floor Clinical Sciences Building, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The presence of lactoferrin at the concentration found in cystic fibrosis (CF) sputum (0.9 g/L) reduced MICs and MBCs of doxycycline for Burkholderia cepacia and Pseudomonas aeruginosa strains. MICs for B. cepacia fell by 32- to 64-fold, from highly resistant to clinically achievable values. Rifampicin MICs for B. cepacia strains were reduced by lactoferrin and for some strains MBCs were reduced. These findings suggest new therapeutic approaches to infections and question the relevance of standard sensitivity tests for CF pathogens. Addition of lactoferrin to media for the routine sensitivity testing of CF isolates might give more relevant results.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colonization of the respiratory tract of cystic fibrosis (CF) patients by mucoid Pseudomonas aeruginosa leads to reduced pulmonary function1 and shorter survival. In P. aeruginosa infections of CF patients, antibiotics that kill the bacteria in vitro fail to eradicate them from the patient. Treatment with antibiotics including doxycycline (Empey, D. & Wood, C., personal communication), to which the bacteria are apparently resistant in vitro, may be associated with clinical improvement. In P. aeruginosa infection in patients other than those with CF, rifampicin (to which the organisms were insensitive in vitro) appeared to confer benefit when it was given in combination with other drugs.2

An important recent development in CF patients has been the emergence of infection by antibiotic resistant Burkholderia cepacia, which is frequently associated with rapid clinical deterioration and sometimes bacteraemia.3 Antibiotic treatment remains unsatisfactory for chronic infections, although it relieves the symptoms of acute pulmonary exacerbation and thus acts as a palliative and not a cure.

Rifampicin is not usually used for the treatment of B. cepacia in CF patients, but rifampicin in combination with other anti-pseudomonal antibiotics was synergic in vitro against B. cepacia isolated from the sputa of CF patients.4 Doxycycline is a semi-synthetically produced substance (6-deoxy-hydroxytetracycline) with similar activity to tetracycline but improved pharmacokinetic properties that permit od dosing, increasing its convenience in clinical use. We know of no reports of its use in the treatment of B. cepacia infections.

Lactoferrin serves as a non-specific antimicrobial agent, which protects against systemic and secretory surface infections.5 Its presence increases the susceptibility of Salmonella typhimurium to rifampicin, ampicillin, ciprofloxacin, erythromycin and chloramphenicol,6 and Escherichia coli to rifampicin.7 Lactoferrin concentration increases during bacterial infection and is found in a very high concentration (0.9 g/L) in the sputa of CF patients colonized with P. aeruginosa.8 This high concentration of lactoferrin increases the susceptiblity of P. aeruginosa9 to rifampicin and chloramphenicol. In view of these findings and the apparent benefit conferred by doxycycline therapy of CF patients with P. aeruginosa infection, we have investigated the effect of lactoferrin at the concentration found in CF sputum on MICs and MBCs of doxycycline for B. cepaciaand P. aeruginosa, and of rifampicin for B. cepacia.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Four mucoid clinical P. aeruginosa isolates were obtained from different CF patients at Withington Hospital, Manchester, UK. All were identified as P. aeruginosa by the API 20 NE system. Nine CF strains of B. cepaciafrom the Central Public Health Laboratory (London, UK) were kindly provided by Fiona Clode, who had identified them as unique strains. Two, 928 and 1146 were of ribotype 1 (the UK epidemic strain) but differed in that 928 was asaccharolytic. IsoSensitest broth (Oxoid, Basingstoke, UK) was used for all MIC and MBC determinations.

The rifampicin and doxycycline powders used (Sigma Ltd, Poole, UK) were 95% and 98% potent, respectively. Other materials and methods were those described previously,9 using the broth microdilution method,10 modified in that an equal volume of organism suspension was added to each working dilution of antibiotic. The inoculum was diluted to give a final concentration in the wells of 104–105/mL. Antibiotic-free controls with and without lactoferrin were included. Human recombinant lactoferrin from Aspergillus awamori was provided by Agennix Inc., Houston, TX, USA. Statistical analysis was by Mann–Whitney U test (two tailed).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All four strains of B. cepacia studied were resistant to doxycycline on conventional MIC and MBC tests (Table I). In the presence of lactoferrin (0.9 g/L), MICs for all strains were significantly lower (32- to 64-fold) for the two sets of eight replicate tests carried out on each strain. MBCs for all strains also fell significantly in the presence of lactoferrin. Heavy growth occurred in the antibiotic-free lactoferrin-containing control. These experiments were repeated with similar results. The P. aeruginosa clinical isolates were similarly resistant to doxycycline, but in the presence of lactoferrin MICs were significantly lower for all strains. MBCs for three of the four strains tested were significantly lower in the presence of lactoferrin.


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Table I. Median (of eight replicates) MICs and MBCs (mg/L) of doxycycline for clinical isolates of B. cepacia and P. aeruginosa with and without 0.9 g/L lactoferrin
 
All nine strains of B. cepacia studied were resistant to rifampicin on conventional MIC and MBC tests (Table II). However, all but one of the strains gave significantly lower MIC values in the presence of lactoferrin. Without lactoferrin, MBCs were above the range studied (>128) for most strains; small reductions in MBC in the presence of lactoferrin were seen for five strains, and were significant for four of them.


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Table II. Median (of eight replicates) MICs and MBCs (mg/L) of rifampicin for clinical strains of B. cepacia with and without 0.9 g/L lactoferrin
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The increased sensitivity of bacteria to antibiotics in the presence of lactoferrin has been reported previously,6,7,9 but the 64-fold fall in MIC for B. cepaciais the largest yet reported for a CF isolate. In most cases in our study, MBCs and MICs fell in the presence of lactoferrin. Such a finding would be unlikely if the effects were solely due to lactoferrin binding iron in the medium, as this would be expected to retard growth rather than kill. The effects we report are thus more likely to be due to other effects of lactoferrin, possibly its ability to damage the Gram-negative outer membrane.7

Our study arose in part from the clinical impression of benefit conferred by doxycycline therapy in CF patients with respiratory infection by P. aeruginosa. The in-vitro effect of lactoferrin in reducing the MICs of doxycycline for P. aeruginosa points to a possible basis for the impression, although MICs of our strains in the presence of lactoferrin were still quite high. The far greater effect on MICs of doxycycline for B. cepacia, an important pathogen for adult CF patients, points to a potentially important new approach to treatment and the necessity for a controlled clinical trial.

The effect of lactoferrin on susceptibility to rifampicin, similar to that already described for P. aeruginosa9 points to its potential for treating B. cepacia in adults with CF. If the results of controlled clinical trials are found to support our findings, this would suggest a need to revise sensitivity tests to match them to in-vivo systems by the addition of lactoferrin to the medium.


    Acknowledgments
 
We would like to thank Dr D. Empey and Professor C. Wood for communication of their impression of the efficacy of doxycycline in CF patients, and Mrs F. Clode, Mr H. Sutcliffe, Mr M. Howard, Professor J. F. Soothill and Mrs M. Thompson for their help.


    Notes
 
* Corresponding author. Tel: +44-161-276-8830; Fax: +44-161-276-8826; E-mail: James.Soothill{at}man.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Pitcher-Wilmot, R. W, Levinsky, R. J., Gordin, I., Turner, M. W. & Matthew, D. J. (1982). Circulating soluble immune complexes containing Pseudomonas antigens in CF. Archives of Disease in Childhood 57, 577–81.[Abstract]

2 . Korvick, J. A., Peacock, J. E., Muder, R. R., Wheeler, R. R. & Yu, V. L. (1992). Addition of rifampicin to antibiotic therapy for Pseudomonas aeruginosa bacteraemia: prospective trial using the zelen protocol. Antimicrobial Agents and Chemotherapy 36,620 –5.[Abstract]

3 . Govan, J. R. W., Hughes, J. E. & Vandame P. (1996). Burkholderia cepacia: medical, taxonomic and ecological issues. Journal of Medical Microbiology45 , 395–407.[Abstract]

4 . Kumar, A., Wofford-McQueen, R. & Gordon, C. (1989). Ciprofloxacin, imipenem and rifampicin: in vitro synergy of two and three drug combinations against Pseudomonas cepacia. Journal of Antimicrobial Chemotherapy 23, 831–5.[Abstract]

5 . Bullen, J. J. (1981). The significance of iron in infection. Reviews of Infectious Diseases 3, 1127–38.[ISI][Medline]

6 . Naidu, A. S. & Arnold, R. R. (1994). Lactoferrin interaction with Salmonellae potentiates antibiotic susceptibility in vitro. Diagnostic Microbiology and Infectious Diseases 20, 69–75.[ISI][Medline]

7 . Ellison, R. T., La Force, M., Ghiel, T. J., Boose, D. S. & Dunn, B. E. (1990). Lactoferrin and transferrin damage of the Gram-negative outer membrane is modulated by Ca2+ and Mg2+. Journal of General Microbiology 136, 1437–46.[ISI][Medline]

8 . Jacquot, J., Tournier, J. M., Carmona, T. G., Puchelle, E., Chazalette, J. P. & Sadoul, E. (1983). Proteines des secretions bronchiques dans la mucoviscidose. Role de l'infection. Bulletin Europeen de Physiopathologie Respiratoire 19, 453–8.[ISI][Medline]

9 . Fowler, C. E., Soothill, J. S. & Oakes, L. (1997). MICs of rifampicin and chloramphenicol for mucoid Pseudomonas aeruginosa strains are lower when human lactoferrin is present. Journal of Antimicrobial Chemotherapy 40, 877–9.[Abstract]

10 . Working Party of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27Suppl. D.

Received 19 November 1998; returned 16 March 1999; revised 29 March 1999; accepted 27 April 1999