Jul 5, 1984 - Effect of Subinhibitory Concentrations of Cephalosporins on Surface. Properties and Siderophore Production in Iron-Depleted Klebsiella.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1985, p. 220-223
Vol. 27, No. 2
0066-4804/85/020220-04$02.00/0 Copyright © 1985, American Society for Microbiology
Effect of Subinhibitory Concentrations of Cephalosporins on Surface Properties and Siderophore Production in Iron-Depleted Klebsiella pneumoniae JAGATH L. KADURUGAMUWA,' HOSMIN ANWAR,' MICHAEL R. W.
BROWN,'* AND OTO ZAK2
Microbiology Research Group, Department of Pharmacy, University of Aston in Birmingham, Birmingham B4 7ET, United Kingdom,' and Pharma Research, Infectious Diseases, Ciba Geigy Ltd., CH-4002 Basel, Switzerland3 Received 5 July 1984/Accepted 26 November 1984
Subinhibitory MICs (sub-MICs) of several cephalosporins significantly reduced the enterochelin production of Klebsiella pneumoniae 327 grown under iron-depleted conditions and also reduced capsule formation regardless of iron availability. The surface hydrophobicity of K. pneumoniae 327 increased significantly when the bacteria were grown in either iron-sufficient or iron-depleted media in the presence of sub-MICs of all the cephalosporins used in this study. Antisera raised against a non-encapsulated K. pneumoniae strain caused rapid agglutination of K. pneumoniae 327 grown in the presence of sub-MICs of the cephalosporins but no agglutination of the same strain grown in drug-free media. The results indicated that the cephalosporins reduced enterochelin production and also capsule formation to the extent that noncapsular surface antigens were exposed, with possible significant consequences in vivo.
Surface properties of bacteria are known to play an important role in infection (4, 19). The composition of the surface components is greatly influenced by the growth environment (2). Recent studies with materials taken directly from human infections (3) or experimentally infected animals (9, 17) have confirmed early findings (6, 8, 21, 22) that the withholding of iron from invading microorganisms plays an important role in host defense against infection. The ability of a microbe to acquire iron is thus an essential requirement for virulence. To scavenge essential iron from the environment, bacteria synthesize and secrete iron-chelating compounds, known as siderophores (11). Chelated iron is taken into the cells by way of high-molecular-weight outer membrane protein (OMP) receptors which are produced in response to iron stress (15). Klebsiella species are known to secrete siderophores of the enterochelin (catechol) type (11), and some strains produce hydroxamate compounds (7). Recent studies have shown that subinhibitory MICs (subMICs) of certain antibiotics can alter the morphology and virulence of bacterial species (12, 18). Despite the known effects of iron depletion and sub-MICs of antibiotics, few workers have taken these factors into account in their in vitro studies. In this initial study, we investigated the effects of sub-MICs of several cephalosporins on the OMP profiles, surface hydrophobicity, and exopolysaccharide and siderophore production in K. pneumoniae under iron-depleted conditions.
100 ion-exhange resin (Bio-Rad Laboratories, Watford, United Kingdom) to remove iron. Iron-sufficient chemically defined medium (Fe+CDM) was the same as Fe-CDM except that 0.025 mM FeSO4 was added. In addition, a micronutrient solution containing the following (final concentrations) was added to both: CaC12, 5 x 10-7 M; HBO3, 5 X 10-7 M; CoCl2, 5 x 10-8 M; CUSO4, 10-8 M; ZnSO4, 10-8 M; MnSO4, 10-7 M; (NH4)6Mo7024, 5 x 10-9 M; and thiamine, 0.025 p,g/ml. Growth. An overnight culture in Fe-CDM was used as an inoculum. Growth was accomplished by shaking 2-liter volumes of the culture in 5-liter Erlenmeyer flasks in an orbital shaker at 37°C. Growth was monitored spectrophotometrically at 470 nm. Glassware. Glassware was washed in 5% (vol/vol) Extran 300 (BDH, Poole, United Kingdom) and then rinsed successively in distilled water, in 1% (vol/vol) HCI, and six times in double-distilled water. Antibiotics. CGP 17520 (synthesized at Ciba-Geigy Ltd., Basel, Switzerland, a new cephalosporin antibiotic carrying an a-aminoacid residue in its side chain (Fig. 1), ceftriaxone (Hoffmann-La Roche Ltd. Basel, Switzerland), cefuroxime and cephalexin (Glaxo Laboratories Ltd., Greenford, United Kingdom), and cefotaxime (Roussel Laboratories, Swindon, United Kingdom) were used in this study. Determination of sub-MICs. MICs were determined by tube dilution. The lowest concentration of antibiotic resulting in the complete inhibition of visible growth was taken as the MIC. A series of sub-MICs of antibiotics were made up in 50 ml of both Fe-CDM and Fe+CDM in 250-ml Erlenmeyer flasks and inoculated with an early stationaryphase, iron-restricted culture. The flasks were incubated in a shaking water bath at 37°C at 140 throws min-1. The samples were transferred to 1% (vol/vol) Formalin in normal saline to investigate the morphological changes taking place during growth. The concentration which had no effect on the growth rate, based on optical density measurements at 470 nm, and which induced filament formation, as vizualised by light microscopy, was chosen as the sub-MIC of the particular antibiotic. A drug-free culture served as the control.
MATERIALS AND METHODS Bacteria. K. pneumoniae 327 (Ciba-Geigy, Basel, Switzerland, culture collection) was used. Media. Cells were grown under iron-sufficient and iron-depleted conditions. Iron-depleted chemically defined medium (Fe-CDM) consisted of 35 mM glucose, 25 mM NH4CI, 1.5 mM KCI, 0.4 mM MgSO4, 0.045 mM NaCl, and 66.66 mM Na2HPO4-NaH2PO4 (pH 7.4). The phosphate buffer had previously been passed twice through a column of Chelex*
Corresponding author. 220
CEPHALOSPORINS AND VIRULENCE FACTORS OF K. PNEUMONIAE
VOL. 27, 1985 OH
HOOCCHCH20CONH
/
-HCONH
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RESULTS AND DISCUSSION s
NH2
N )> C H2S
N-N N
COO' N+'Na CH3
FIG. 1. Formula of CGP 17520.
Assay of siderophores in culture supernatants. The method used to detect enterochelin was based on that described by Arnow (1), and hydroxamate siderophores were detected by the method of Holzberg and Artis (10). An Escherichia coli ColV+ strain (kindly provided by E. Griffiths) which is known to produce hydroxamate siderophores was used as a positive control. Surface hydrophobicity. Essentially, the contact angles of drops of saline were measured by using a cellulose-acetate membrane filter layered with washed bacteria from a 20-ml, optical density 10 culture as described by van Oss (20). Preparation of outer membranes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Outer membranes were prepared with sodium N-lauroyl sarcosinate (Sarkosyl; Sigma Chemical Co., Poole, United Kingdom) as described by Filip et al. (5), and the OMP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Lugtenberg et al. (13) with 10% acrylamide gels and purified sodium dodecyl sulfate (99% pure, specially purified; BDH, Poole, United Kingdom).
The MICs of CGP 17520, ceftriaxone, cephalexin, cefuroxime, and cefotaxime for K. pneumoniae 327 were 2.68, 0.02, 2.5, 1.25, and 0.016 jig/ml, respectively. The concentrations of the antibiotics used in this study were 10, 20, 33, 33, and 33% of the MICs, respectively. Figure 2 shows the OMP profiles of K. pneumoniae 327 during growth under iron-sufficient and iron-depleted conditions in the absence of any antibiotics. A number of highmolecular-weight protein bands were induced under iron-depleted conditions (lanes 6 through 10). The Mrs of these proteins were 83,000 (83k), 78k, 75k, 73k, 70k, and 69k. The induction of high-molecular-weight OMP under iron-depleted conditions has been observed in other species, such as Pseudomonas aeruginosa (3) and E. coli (9). They have been suggested to act as receptors for iron-siderophore complexes in high-affinity iron uptake systems. The 48k Mr protein was strongly expressed in the outer membranes of cells grown in iron-sufficient medium (lanes 1 through 5) but was significantly reduced in the outer membranes of cells grown in iron-depleted medium (lanes 6 through 10). This protein may function as a receptor for a low-affinity, membrane-bound iron uptake system. The OMP patterns in the presence of sub-MICs of the cephalosporins appeared identical to those in the absence of any antibiotics except that there was an indication that the iron-regulated membrane proteins were expressed earlier in the growth curve (data not shown). Figure 3 illustrates enterochelin production during growth in Fe+CDM and Fe-CDM in the presence or absence of sub-MICs of cephalosporins. A general conclusion that the production of enterochelin was reduced by half in the presence of sub-MICs of cephalosporins in iron-depleted medium can be drawn. Enterochelin was barely detectable in the supernatants of the iron-sufficient cultures. Entero-
16
14 12
FIG. 2. OMP profiles of K. pneumoniae 327 during growth under iron-sufficient and iron-depleted conditions. Lanes: 1, Fe+CDM, optical density of 0.8; 2, Fe+CDM, optical density of 2.5; 3, Fe+CDM, optical density of 4.9; 4, Fe+CDM, optical density of 6.2; 5, Fe+CDM, optical density of 6.7; 6, Fe-CDM, optical density of 0.8; 7, Fe-CDM, optical density of 2.3; 8, Fe-CDM, optical density of 3.4; 9, Fe-CDM, optical density of 4.7; 10, Fe-CDM, optical density of 5.2. A 250-ml portion of each culture was removed from a 5-liter culture flask (see text) at 60-min intervals for outer membrane preparation. Fe+CDM cultures started to enter the stationary phase at an optical density of 5.8, and Fe-CDM cultures did so at an optical density of 5.3. Identical results were obtained for cells grown in the presence of sub-MICs of cephalosporins. The numbers at the right indicate molecular weights in thousands.
-1
5
6
7
8
9
10
HOURS FIG. 3. Effect of sub-MICs of cephalosporins on enterochelin production by K. pneumoniae 327. Symbols: E, Fe-CDM; O, Fe-CDM plus sub-MIC of a cephalosporin; 0, Fe+CDM; 0, Fe+CDM plus sub-MIC of a cephalosporin. The highest concentration of a cephalosporin (see text) not affecting the growth rate was added to Fe+CDM and Fe-CDM, and enterochelin production was monitored during batch growth. Similar results were obtained for all cephalosporins (cephalexin is shown).
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KADURUGAMUWA ET AL.
chelin production is essential for the growth of bacteria under iron-depleted conditions. It enables the organism to scavenge iron present in the growth environment and transport it via the receptors located in the outer membrane into the interior of the bacterial cells (11). The production of enterochelin has been demonstrated in vivo in members of the family Enterobacteriaceae (8), and others have reported that the host immune system responds by synthesizing specific antibodies against the siderophores (14). Hydroxamate siderophore production was also investigated in this study. However, we were unable to detect any hydroxamate production in the supernatants of any of the cultures used in this study. Similar observations have previously been reported (16). Cultures grown in the presence of sub-MICs of all the cephalosporins invariably lost their characteristic gel-like viscosity, and microscopic examinations, in the presence of India ink confirmed a reduction in capsules. To test whether the reduction in capsules exposed otherwise masked antigenic sites, antiserum was raised against a non-encapsulated mutant of K. pneumoniae NCTC 5055 (23). A slide agglutination assay was used to determine whether cells grown in the presence of sub-MICs of the three cephalosporins most effective in reducing culture viscosity and capsule production (CGP 17520, ceftriaxone, and cefotaxime) would react differently from those grown in the absence of cephalosporins. Agglutination did not occur with cells grown in the absence of cephalosporins, whereas very rapid agglutination was observed with cells grown in the presence of sub-MICs of all three cephalosporins. This indicates that sub-MICs of cephalosporins resulted in the exposure of outer membrane antigens which are otherwise masked. Table 1 shows the contact angles of K. pneumoniae 327 grown under iron-sufficient or iron-depleted conditions in the presence or absence of sub-MICs of cephalosporins. The contact angles of the control cells grown in either ironsufficient medium or iron-depleted medium, were very hydrophilic (110). Growth of the organism in the presence of sub-MICs of cephalosporins significantly increased the conTABLE 1. Surface hydrophobicity of K. pneumoniae 327 grown in the presence of sub-MICs of cephalosporins Contact angle Culture condition (degree) No antibiotics Fe+CDM .................. 11.75 ± 0.71 Fe-CDM .................. 11.81 ± 0.71 CGP 17520 (10% of MIC) Fe+CDM .................. Fe-CDM ..................
26.0 1.45 19.13 ± 0.83
Ceftriaxone (20% of MIC)
Fe+CDM ................... 21.75 ± 0.96 Fe-CDM .................. 20.11 ± 0.92
Cephalexin (33% of MIC) Fe+CDM ................... Fe-CDM ..................
17.33 ± 0.82 17.75 ± 0.90
Cefuroxime (33% of MIC) Fe+CDM .................. Fe-CDM ..................
17.33 ± 0.82 17.95 ± 0.98
Cefotaxime (33% of MIC) Fe+CDM ................... Fe-CDM ..................
18.33 ± 0.75 18.10 ± 0.26
tact angles to 17 to 26°. The increase in the hydrophobicity of the surfaces of the cephalosporin-induced filaments again indicates the exposure of otherwise masked components. We also used hydrophobic interaction chromatography (24) to investigate the hydrophobicity of the surfaces of bacteria. However, we found that this method cannot be applied to filamentous bacteria, as the percentage of filamentous bacteria adsorbed onto Sepharose CL-4B (control) was similar to that adsorbed onto phenyl-Sepharose. This may be due to the trapping of filamentous cells on the column, and we suggest that this method not be used to determine the hydrophobicity of filamentous cells. In conclusion, this study demonstrated that under iron-depleted conditions, sub-MICs of CGP 17520, ceftriaxone, cephalexin, cefuroxime, and cefotaxime significantly reduced enterochelin production by bacteria without affecting the growth rate. These cephalosporins also increased the hydrophobicity of the cell surface by reducing the amount of capsule produced and exposing other outer membrane components of encapsulated K. pneumoniae 327. The increase in the hydrophobicity of cells grown in the presence of cephalosporins occurred under iron-sufficient and iron-depleted conditions. The exposure of surface antigens by sub-MICs of cephalosporins and the reduction in enterochelin production and capsule formation are novel modes of action. In vivo such effects would reduce the capacity of a bacterium to survive in the host. ACKNOWLEDGMENTS We are grateful to P. A. Lambert, P. Williams, and G. H. Shand for helpful discussions and to E. Griffiths for providing the E. coli Col V+ strain. Part of this work was supported by the Cystic Fibrosis Research Trust of the United Kingdom. LITERATURE CITED 1. Arnow, L. E. 1937. Colorimetric determination of the components of 3,4-dihydroxyphenylalanine-tyrosine mixtures. J. Biol. Chem. 228:531-537. 2. Brown, M. R. W. 1977. Nutrient depletion and antibiotic susceptibility. J. Antimicrob. Chemother. 3:198-201. 3. Brown, M. R. W., H. Anwar, and P. A. Lambert. 1984. Evidence that mucoid Pseudomonas aeruginosa in the cystic fibrosis lung grows under iron restriction. FEMS Microbiol. Lett. 21:113-117. 4. Costerton, J. W., M. R. W. Brown, and J. M. Sturgess. 1979. The envelope: its role in infection, p. 41-62. In R. G. Doggett (ed.), Pseudomonas aeruginosa: clinical manifestation of infection and current therapy. Academic Press, Inc., New York. 5. Filip, C., G. Fletcher, J. L. Wulff, and C. F. Earhart. 1973. Solubilization of the cytoplasmic membrane of Escherichia coli. J. Bacteriol. 115:717-722. 6. Finkelstein, R. A., C. V. Sciortino, and M. A. McIntosh. 1983. Role of iron in microbe-host interaction. Rev. Infect. Dis. 5(Suppl.):759-777. 7. Gibson, F., and D. I. Magrath. 1969. The isolation and characterisation of a hydroxamic acid (aerobactin) formed by Aerobacter aerogenes. Biochim. Biophys. Acta 192:175-184. 8. Griffiths, E. 1983. Availability of iron and survival of bacteria in infection, p. 153-177. In C. S. F. Easmon, J. Jeljaszewicz, M. R. W. Brown, and P. A. Lambert (ed.), Medical microbiology, vol. 3. Academic Press, Inc., London. 9. Griffiths, E., P. Stevenson, and P. Joyce. 1983. Pathogenic Escherichia coli express new outer membrane protein when grown in vivo. FEMS Microbiol. Lett. 16:95-99. 10. Holzberg, M., and W. M. Artis. 1983. Hydroxamate siderophore production in opportunistic and systemic fungal pathogens. Infect. Immun. 40:1134-1139. 11. Lankford, C. E. 1973. Bacterial assimilation of iron. Crit. Rev. Microbiol. 2:273-331.
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