FEMS MicrobiologyLetters 81 (1991) 67-72 © 1991 Federation of European MicrobiologicalSocieties0378-1097/91/$03.50 Published by Elsevier ADONIS 037810979100290N
67
FEMSLE 04486
Susceptibility of iron-loaded Borrelia burgdorferi to killing by hydrogen peroxide and human polymorphonuclear leucocytes Vittorio Sambri, R o b e r t o C e v e n i n i a n d M i c h e l e L a Placa Institute of Microbiology, University of Bologna, S. Orsola Hospital, Bologna, Italy
Received 13 August 1990 Revision received22 February 1991 Accepted 11 March 1991 Key words: Borrelia burgdorferi; Polymorphonuclear leucocyte; Iron; Hydrogen peroxide
1. SUMMARY Borrelia burgdorferi grew more slowly in irondepleted than in iron-sufficient media. The addition of increasing concentrations of iron stimulated borrelial growth and resulted in the intracellular accumulation of this element. Compared with iron-starved borrelia, iron-enriched organisms showed enhanced sensitivity to hydrogen peroxide. Intracellular iron-content did not, however, influence susceptibility to killing by hydrogen peroxide and killing by human polymorphonuclear leucocytes.
the phagocytosis of bacteria. These oxidants react to form highly toxic radicals in the presence of iron, following the Haber-Weiss mechanism [1]. This study aimed to determine whether Borrelia burgdorferi, the etiological agent of Lyme disease [2], was able to concentrate iron when cultured in the presence of increasing concentrations of this substance and whether iron loading rendered borrelia more sensitive to killing by hydrogen peroxide and by human PMNs.
3. MATERIALS A N D M E T H O D S
2. I N T R O D U C T I O N
3.1. Iron loading and starvation Borrelia burgdorferi strain IRS [3] was grown in
Superoxide (O~-) and its disproportionation product hydrogen peroxide (H202) are produced in large amounts by human polymorphonuclear leucocytes (PMNs) when these cells are engaged in
BSK II medium as previously described [4]. For iron-loading experiments bacteria were grown in glass tubes containing 10 ml of sterile ferrous ammonium sulfate (Merck, F.R.G.) (0-2000 '/~M) until the number of viable borreliae in the medium reached 3 x 10 8 ml i as determined by dark-field microscopy. To evaluate whether borrelia could grow in iron-deprived media they were grown in BSK II passed through a Bio Rex Chelex ion
Correspondence to." V. Sambri, Institute of Microbiology,University of Bologna, S. Orsola Hospital, 9 via Massarenti, 40138 Bologna, Italy.
68 exchange membrane (Bio Rad, U.S.A.) to reduce the basal level of iron present in the medium. After growth, bacteria were harvested by centrifugation, washed three times in 0.01 M sodium phosphate buffer containing 0.15 M NaCI, p H 7.2 (PBS) and used for iron content determination or for in vitro evaluation of killing by hydrogen peroxide and killing by human PMNs.
3.2. Determination of bacterial iron content The iron content of bacteria was determined using the phenanthroline assay [5], according to the method described by Hoepelman et al. [6] with minor modifications. Briefly, bacteria were harvested as described above, were counted in dark-field microscopy at 400 x and resuspended in PBS to a concentration of 10 9 ml -~. The bacterial suspension was sonicated to lyse the cells and the following reagents were added: 50 /~1 of 0.6 M ascorbic acid, 1 ml of 1,25-phenanthroline (25 mM). The p H of the solution was brought to 2 using 1 M HC1 and the volume was adjusted to 5 ml by adding sterile, iron-free distilled water. The mixture was left for 30 min at room temperature and centrifuged at 9000 x g for 20 min to precipitate cellular debris, and the supernatant was assayed for iron content. Results for iron concentration were the mean of five determinations.
3.4. Measurement of bacterial uptake and killing by PMNs Phagocytosis and killing by PMNs were studied using the method of Van Furth [7]: live unopsonized borreliae were mixed at a 10:1 ratio with P M N s (2 x 107 m1-1 in GHBSS and incubated at 37°C with shaking at 8 rpm. Samples were taken immediately, and every 30 min up to 2 h of incubation. B a c t e r i a / P M N suspensions were centrifuged at 110 x g for 4 min and a 200-ffl sample was taken to determine the number of viable and replicating microorganisms remaining in the medium after removing the PMNs. Bacterial internalization by P M N s was determined after washing the cells, by immunofluorescence using the method described by Benach et al. [8] and by observation in dark-field microscopy. PMNs were then lysed by resuspension in 1% BSA (Sigma, U.S.A.) in double-distilled water and a sample was cultured in BSK II as described above to determine the number of intraphagocytic viable bacteria. Serial 10-fold dilutions were also incubated at 34°C for 14 days and observed in dark-field microscopy to confirm borrelial viability.
4. R E S U L T S
3.3. Sensitivity to killing by hydrogen peroxide Killing of borreliae by hydrogen peroxide was measured by adding 2 x 10 6 live bacteria to 1 ml of sterile Hanks' balanced salt solution (Gibco, U.K.) containing 0.1% gelatin (GHBSS), to which increasing concentrations of H202 were added (0100 mM). After adding the bacterial suspension each tube was promptly sampled (200/xl; time 0), incubated under sterile conditions in a shaking incubator at 37°C for 30 rain (150 rpm) and then left stationary for an additional 30 rain. At the end of this period (60 min) samples were taken from each tube and diluted 10-fold in BSK II. The highest dilution in which viable borreliae were detectable was defined as the titre of the bacterial culture. Each determination was the mean of five samples from triplicate tubes. In addition, after 14 days incubation each t u b e was sampled and observed using dark-field microscopy to ensure that live borreliae were present.
4.1. Growth of B. burgdorferi in iron-depleted medium Preliminary studies were carried out to assess the iron content of BSK II medium by filtering 100 ml of normal complete BSK II medium through a Bio Rex Chelex ion exchange membrane. The iron content of BSK II medium was calculated as 90 fig 1-1 (1.6 /~M). After passage through the Bio Rex membrane, the medium iron content was below detectable levels. Borreliae were able to grow in this chelexed medium, although cultures of iron-starved borreliae reached a titre of 108 in a period of 2 weeks, in contrast to the 1 week normally required.
4.2. Iron loading of borreliae Whilst borreliae grown in BSK II medium possessed an intracellular iron content of 0.27 fig 10 - 9 bacteria, that of iron-depleted organisms
69
was below detectable levels. Bacteria grown in the presence of increasing ferrous ammonium sulphate concentrations demonstrated varying degrees of iron accumulation. For medium iron concentrations of 500, 1000, 1500 and 2000 # M the relative mean borrelial iron contents were 0.79, 1.18, 2.64 and 2.97 #g 10 -9 organisms, respectively. This rise in bacterial iron concentration was only evident after disruption of bacterial cells and suggests that the accumulated iron was located intracellularly.
drogen peroxide, with respect to BSK II grown bacteria. A concentration of 0.1 mM H 2 0 2 that did not kill BSK II grown borreliae after exposure for 60 rain caused at least 106-fold reduction in the titre of borreliae grown in the presence of excess iron. Exogenous catalase also proved to be active in protecting these iron-loaded organisms. The sensitivity of iron-starved bacteria to killing by hydrogen peroxide, however, was similar to that demonstrated by BSK II grown borreliae.
4.3. In vitro sensitivity of BSK H grown, iron-loaded and iron-starved borreliae to hydrogen peroxide The results summarized in Table 1 show that borreliae are highly susceptible to hydrogen peroxide. An almost immediate reduction in the number of viable bacteria was observed using H202 at concentrations from 1.0 to 100 raM. After 60 min of incubation the titre of borreliae grown in BSK II medium and exposed to as little as 1.0 mM H 2 0 2 w a s reduced from 10 -7 to 10 - 2 . The addition of catalase from bovine liver (1.100 U / m l ) to the incubation medium completely inhibited the action of H202 on borreliae, suggesting that their susceptibility to this oxidizing factor probably involves hydroxyl radicals. Increasing intraceUular iron contents enhanced killing by hy-
4.4. Phagocytosis and killing of B S K H grown, iron-loaded and iron-deprived B. burgdorferi The internalization of B. burgdorferi was studied by immunofluorescence microscopy, using the method described by Benach et al. [8]. After 2 h of incubation no extracellular bacterial bodies were detectable, whereas cells containing intracytoplasmic spirochaetes or fluorescent bodies were present (Fig. 1). No differences were observed in bacterial uptake of normal, iron-deprived and iron-rich borreliae by human PMNs. After 2 h of incubation in the presence of PMNs, the extracellular iron-loaded, iron-deprived or BSK II grown bacteria were cultured. After 2 weeks the titre was determined: all the tubes were positive up to a dilution of 10 -8. Similarly, no variation was ob-
Table 1 Sensitivity of B. burgdorferi to H202 when grown in BSK II medium containing increasing iron concentrations at times 0 and 60 min H 202 concentration (raM) 100 10 1
Ferrous ammonium sulfate concentration (#M) 0 1 x 10 -3
0
1 x 10 -2
1000 1 X 10 -2
1500 1 X 10 -2
2 000 l X 10 -2
(_) ~,b
(_)
(_)
(_)
(_)
1 X 10 -7 (1 x 10- ~) 1 × 1 0 -v
1 X 10 -6 (-) 1 x 1 0 -5 (] x lO-~) l x l 0 -6 (1 x 10 -1 ) 1 ×10 - s (1 X 10 -7)
1 X 10 -6 (-) l x 1 0 -5
1 X 10 -5 (-) l x 1 0 -5
1 X 10 -3 (-) l x 1 0 -3
(-)
(-)
(-)
l x l 0 -5 (-) lxl0 -s (1 x 10 - s )
l x l 0 -5 (-) 1 x l 0 -8 (1 x 10 -8)
1 x l 0 -3 (-) 1 x l 0 -8 (1 x 10 - s )
(1 x 10-2) 0.1
500
l X l 0 -8 (1 x 10 -7) 1 x l 0 -8 (1 X 10 - s )
The infectivity titre was taken as the highest dilution containing viable organisms, by dark-field microscopy. a In brackets: titres at 60 min. b --, no viable bacteria detectable.
70
Fig. 1. Human polymorphonuclear leucocytes after 2 h incubation with B. burgdorferi and three washings in PBS, as detected by immunofluorescence performed with guinea pig immune serum. No extracellular bacterial bodies are present whereas cytoplasmic reactivity is clearly evident.
served in the number of intraphagocytic viable bacteria determined by culture for normally grown and iron-rich or iron-starved B. burgdorferi.
5. D I S C U S S I O N Bacterial virulence is known to be greatly influenced by iron and many bacteria have developed a variety of systems to extract this fundamental element from their environment [9]. In addition some authors have reported that hyperferremia may increase susceptibility to certain pathogens [10], whilst others have noted that a fall in plasma iron content is a common finding during several episodes of certain infectious diseases [9]. Bearing these reports in mind, we examined the influence of iron on the growth and susceptibility of B. burgdorferi to killing by hydrogen peroxide and by PMNs. Our findings demonstrated that B. burgdorferi, like other microorganisms [6], is able to accumulate iron when grown in increasing quantities of this element and
that the amount of intracellular iron is related to the growth medium iron content. Staphylococcus aureus, like iron-rich borreliae, are also known to be more sensitive to killing by hydrogen peroxide in vitro [6]. This finding correlates well with the recent data presented by J.P. Padgett and D.J. Pekala (IV Int. Conf. Lyme Borreliosis, Stockholm, 18-21 June 1990, personal communication) showing an almost total absence of intrinsic catalase activity in B. burgdorferi. In addition, the uptake of B. burgdorferi by human PMNs was not influenced by alterations in the intrinsic iron content, nor did the intracellular iron concentration affect B. burgdorferi susceptibility to kilting by human PMNs. These findings are in agreement with results obtained by Repine et al. [11] and Hoepelman et al. [6] who showed that increased intracelhilar concentration did not alter the susceptibility of S. aureus to kilting by PMNs. Differences between the kilting rate of iron-loaded and control bacteria may therefore conceivably have been masked by the multiple bactericidal mechanisms operating within normal PMNs.
71 ACKNOWLEDGEMENTS We are i n d e b t e d to Mr. Enzo Della Bella for his c o n t r i b u t i o n in chemical assays, to Miss Francesca Massaria for excellent technical work a n d to G. Sermasi, M D (S. Orsola Hospital, C e n t r a l Blood Bank) for p r o v i d i n g h u m a n P M N s . We are also i n d e b t e d to Dr. A l e s s a n d r o Ripalti for his helpful c o n t r i b u t i o n .
REFERENCES [1] Babior, B.M. (1984) Blood 64, 959-966. [2] Burgdorfer, W., Barbour, A.G., Hayes, S.F., Benach, J.L., Grunwald, E. and Davis, J.P. (1982) Science 216, 13171319.
[3] Barbour, A.G., Burgdorfer, W., Hayes, S.F., Peter, O. and Aeschlimann, A. (1983) Curr. Microbiol. 8, 123-126. [4] Sambri, V. and Lovett, M.A. (1990) Microbiologica 13, 79-83. [5] Iron Sub-committee (1978) Analyst 103, 391-396. [6] Hoepelman, I.M., Bezemer, W.A., Vandenbroucke-Grauls, C.M., Marx, J.J. and Verhoef, J. (1990) Infect. Immun. 58, 26-31. [7] Van Furth, R., Van Zwet, L. and Leijh, P.C.J. (1979) in Cellular Immunology (Weir, D.M., Ed.), 3rd Edn., pp. 32.1-32.19, Blackwell Scientific, Oxford. [8] Benach, J.L., Fleit, H.B., Habicht, G.S., Coleman, J.L., Bosler, E.M. and Lane, B.P. (1984) J. Infect. Dis. 150, 497-507. [9] Brock, J.H. (1986) Br. Med. J. 293, 518-519. [10] Jones, R.L., Peterson, C.M., Grady, R.W., Kumbaraci, T. and Cerami, A. (1977) Nature (London) 26, 63-69. [11] Repine, J.E., Fox, R.B., Berger, E.M. and Harada, R.N. (1981) Infect. Immun. 32, 407-410.
67
FEMSLE 04486
Susceptibility of iron-loaded Borrelia burgdorferi to killing by hydrogen peroxide and human polymorphonuclear leucocytes Vittorio Sambri, R o b e r t o C e v e n i n i a n d M i c h e l e L a Placa Institute of Microbiology, University of Bologna, S. Orsola Hospital, Bologna, Italy
Received 13 August 1990 Revision received22 February 1991 Accepted 11 March 1991 Key words: Borrelia burgdorferi; Polymorphonuclear leucocyte; Iron; Hydrogen peroxide
1. SUMMARY Borrelia burgdorferi grew more slowly in irondepleted than in iron-sufficient media. The addition of increasing concentrations of iron stimulated borrelial growth and resulted in the intracellular accumulation of this element. Compared with iron-starved borrelia, iron-enriched organisms showed enhanced sensitivity to hydrogen peroxide. Intracellular iron-content did not, however, influence susceptibility to killing by hydrogen peroxide and killing by human polymorphonuclear leucocytes.
the phagocytosis of bacteria. These oxidants react to form highly toxic radicals in the presence of iron, following the Haber-Weiss mechanism [1]. This study aimed to determine whether Borrelia burgdorferi, the etiological agent of Lyme disease [2], was able to concentrate iron when cultured in the presence of increasing concentrations of this substance and whether iron loading rendered borrelia more sensitive to killing by hydrogen peroxide and by human PMNs.
3. MATERIALS A N D M E T H O D S
2. I N T R O D U C T I O N
3.1. Iron loading and starvation Borrelia burgdorferi strain IRS [3] was grown in
Superoxide (O~-) and its disproportionation product hydrogen peroxide (H202) are produced in large amounts by human polymorphonuclear leucocytes (PMNs) when these cells are engaged in
BSK II medium as previously described [4]. For iron-loading experiments bacteria were grown in glass tubes containing 10 ml of sterile ferrous ammonium sulfate (Merck, F.R.G.) (0-2000 '/~M) until the number of viable borreliae in the medium reached 3 x 10 8 ml i as determined by dark-field microscopy. To evaluate whether borrelia could grow in iron-deprived media they were grown in BSK II passed through a Bio Rex Chelex ion
Correspondence to." V. Sambri, Institute of Microbiology,University of Bologna, S. Orsola Hospital, 9 via Massarenti, 40138 Bologna, Italy.
68 exchange membrane (Bio Rad, U.S.A.) to reduce the basal level of iron present in the medium. After growth, bacteria were harvested by centrifugation, washed three times in 0.01 M sodium phosphate buffer containing 0.15 M NaCI, p H 7.2 (PBS) and used for iron content determination or for in vitro evaluation of killing by hydrogen peroxide and killing by human PMNs.
3.2. Determination of bacterial iron content The iron content of bacteria was determined using the phenanthroline assay [5], according to the method described by Hoepelman et al. [6] with minor modifications. Briefly, bacteria were harvested as described above, were counted in dark-field microscopy at 400 x and resuspended in PBS to a concentration of 10 9 ml -~. The bacterial suspension was sonicated to lyse the cells and the following reagents were added: 50 /~1 of 0.6 M ascorbic acid, 1 ml of 1,25-phenanthroline (25 mM). The p H of the solution was brought to 2 using 1 M HC1 and the volume was adjusted to 5 ml by adding sterile, iron-free distilled water. The mixture was left for 30 min at room temperature and centrifuged at 9000 x g for 20 min to precipitate cellular debris, and the supernatant was assayed for iron content. Results for iron concentration were the mean of five determinations.
3.4. Measurement of bacterial uptake and killing by PMNs Phagocytosis and killing by PMNs were studied using the method of Van Furth [7]: live unopsonized borreliae were mixed at a 10:1 ratio with P M N s (2 x 107 m1-1 in GHBSS and incubated at 37°C with shaking at 8 rpm. Samples were taken immediately, and every 30 min up to 2 h of incubation. B a c t e r i a / P M N suspensions were centrifuged at 110 x g for 4 min and a 200-ffl sample was taken to determine the number of viable and replicating microorganisms remaining in the medium after removing the PMNs. Bacterial internalization by P M N s was determined after washing the cells, by immunofluorescence using the method described by Benach et al. [8] and by observation in dark-field microscopy. PMNs were then lysed by resuspension in 1% BSA (Sigma, U.S.A.) in double-distilled water and a sample was cultured in BSK II as described above to determine the number of intraphagocytic viable bacteria. Serial 10-fold dilutions were also incubated at 34°C for 14 days and observed in dark-field microscopy to confirm borrelial viability.
4. R E S U L T S
3.3. Sensitivity to killing by hydrogen peroxide Killing of borreliae by hydrogen peroxide was measured by adding 2 x 10 6 live bacteria to 1 ml of sterile Hanks' balanced salt solution (Gibco, U.K.) containing 0.1% gelatin (GHBSS), to which increasing concentrations of H202 were added (0100 mM). After adding the bacterial suspension each tube was promptly sampled (200/xl; time 0), incubated under sterile conditions in a shaking incubator at 37°C for 30 rain (150 rpm) and then left stationary for an additional 30 rain. At the end of this period (60 min) samples were taken from each tube and diluted 10-fold in BSK II. The highest dilution in which viable borreliae were detectable was defined as the titre of the bacterial culture. Each determination was the mean of five samples from triplicate tubes. In addition, after 14 days incubation each t u b e was sampled and observed using dark-field microscopy to ensure that live borreliae were present.
4.1. Growth of B. burgdorferi in iron-depleted medium Preliminary studies were carried out to assess the iron content of BSK II medium by filtering 100 ml of normal complete BSK II medium through a Bio Rex Chelex ion exchange membrane. The iron content of BSK II medium was calculated as 90 fig 1-1 (1.6 /~M). After passage through the Bio Rex membrane, the medium iron content was below detectable levels. Borreliae were able to grow in this chelexed medium, although cultures of iron-starved borreliae reached a titre of 108 in a period of 2 weeks, in contrast to the 1 week normally required.
4.2. Iron loading of borreliae Whilst borreliae grown in BSK II medium possessed an intracellular iron content of 0.27 fig 10 - 9 bacteria, that of iron-depleted organisms
69
was below detectable levels. Bacteria grown in the presence of increasing ferrous ammonium sulphate concentrations demonstrated varying degrees of iron accumulation. For medium iron concentrations of 500, 1000, 1500 and 2000 # M the relative mean borrelial iron contents were 0.79, 1.18, 2.64 and 2.97 #g 10 -9 organisms, respectively. This rise in bacterial iron concentration was only evident after disruption of bacterial cells and suggests that the accumulated iron was located intracellularly.
drogen peroxide, with respect to BSK II grown bacteria. A concentration of 0.1 mM H 2 0 2 that did not kill BSK II grown borreliae after exposure for 60 rain caused at least 106-fold reduction in the titre of borreliae grown in the presence of excess iron. Exogenous catalase also proved to be active in protecting these iron-loaded organisms. The sensitivity of iron-starved bacteria to killing by hydrogen peroxide, however, was similar to that demonstrated by BSK II grown borreliae.
4.3. In vitro sensitivity of BSK H grown, iron-loaded and iron-starved borreliae to hydrogen peroxide The results summarized in Table 1 show that borreliae are highly susceptible to hydrogen peroxide. An almost immediate reduction in the number of viable bacteria was observed using H202 at concentrations from 1.0 to 100 raM. After 60 min of incubation the titre of borreliae grown in BSK II medium and exposed to as little as 1.0 mM H 2 0 2 w a s reduced from 10 -7 to 10 - 2 . The addition of catalase from bovine liver (1.100 U / m l ) to the incubation medium completely inhibited the action of H202 on borreliae, suggesting that their susceptibility to this oxidizing factor probably involves hydroxyl radicals. Increasing intraceUular iron contents enhanced killing by hy-
4.4. Phagocytosis and killing of B S K H grown, iron-loaded and iron-deprived B. burgdorferi The internalization of B. burgdorferi was studied by immunofluorescence microscopy, using the method described by Benach et al. [8]. After 2 h of incubation no extracellular bacterial bodies were detectable, whereas cells containing intracytoplasmic spirochaetes or fluorescent bodies were present (Fig. 1). No differences were observed in bacterial uptake of normal, iron-deprived and iron-rich borreliae by human PMNs. After 2 h of incubation in the presence of PMNs, the extracellular iron-loaded, iron-deprived or BSK II grown bacteria were cultured. After 2 weeks the titre was determined: all the tubes were positive up to a dilution of 10 -8. Similarly, no variation was ob-
Table 1 Sensitivity of B. burgdorferi to H202 when grown in BSK II medium containing increasing iron concentrations at times 0 and 60 min H 202 concentration (raM) 100 10 1
Ferrous ammonium sulfate concentration (#M) 0 1 x 10 -3
0
1 x 10 -2
1000 1 X 10 -2
1500 1 X 10 -2
2 000 l X 10 -2
(_) ~,b
(_)
(_)
(_)
(_)
1 X 10 -7 (1 x 10- ~) 1 × 1 0 -v
1 X 10 -6 (-) 1 x 1 0 -5 (] x lO-~) l x l 0 -6 (1 x 10 -1 ) 1 ×10 - s (1 X 10 -7)
1 X 10 -6 (-) l x 1 0 -5
1 X 10 -5 (-) l x 1 0 -5
1 X 10 -3 (-) l x 1 0 -3
(-)
(-)
(-)
l x l 0 -5 (-) lxl0 -s (1 x 10 - s )
l x l 0 -5 (-) 1 x l 0 -8 (1 x 10 -8)
1 x l 0 -3 (-) 1 x l 0 -8 (1 x 10 - s )
(1 x 10-2) 0.1
500
l X l 0 -8 (1 x 10 -7) 1 x l 0 -8 (1 X 10 - s )
The infectivity titre was taken as the highest dilution containing viable organisms, by dark-field microscopy. a In brackets: titres at 60 min. b --, no viable bacteria detectable.
70
Fig. 1. Human polymorphonuclear leucocytes after 2 h incubation with B. burgdorferi and three washings in PBS, as detected by immunofluorescence performed with guinea pig immune serum. No extracellular bacterial bodies are present whereas cytoplasmic reactivity is clearly evident.
served in the number of intraphagocytic viable bacteria determined by culture for normally grown and iron-rich or iron-starved B. burgdorferi.
5. D I S C U S S I O N Bacterial virulence is known to be greatly influenced by iron and many bacteria have developed a variety of systems to extract this fundamental element from their environment [9]. In addition some authors have reported that hyperferremia may increase susceptibility to certain pathogens [10], whilst others have noted that a fall in plasma iron content is a common finding during several episodes of certain infectious diseases [9]. Bearing these reports in mind, we examined the influence of iron on the growth and susceptibility of B. burgdorferi to killing by hydrogen peroxide and by PMNs. Our findings demonstrated that B. burgdorferi, like other microorganisms [6], is able to accumulate iron when grown in increasing quantities of this element and
that the amount of intracellular iron is related to the growth medium iron content. Staphylococcus aureus, like iron-rich borreliae, are also known to be more sensitive to killing by hydrogen peroxide in vitro [6]. This finding correlates well with the recent data presented by J.P. Padgett and D.J. Pekala (IV Int. Conf. Lyme Borreliosis, Stockholm, 18-21 June 1990, personal communication) showing an almost total absence of intrinsic catalase activity in B. burgdorferi. In addition, the uptake of B. burgdorferi by human PMNs was not influenced by alterations in the intrinsic iron content, nor did the intracellular iron concentration affect B. burgdorferi susceptibility to kilting by human PMNs. These findings are in agreement with results obtained by Repine et al. [11] and Hoepelman et al. [6] who showed that increased intracelhilar concentration did not alter the susceptibility of S. aureus to kilting by PMNs. Differences between the kilting rate of iron-loaded and control bacteria may therefore conceivably have been masked by the multiple bactericidal mechanisms operating within normal PMNs.
71 ACKNOWLEDGEMENTS We are i n d e b t e d to Mr. Enzo Della Bella for his c o n t r i b u t i o n in chemical assays, to Miss Francesca Massaria for excellent technical work a n d to G. Sermasi, M D (S. Orsola Hospital, C e n t r a l Blood Bank) for p r o v i d i n g h u m a n P M N s . We are also i n d e b t e d to Dr. A l e s s a n d r o Ripalti for his helpful c o n t r i b u t i o n .
REFERENCES [1] Babior, B.M. (1984) Blood 64, 959-966. [2] Burgdorfer, W., Barbour, A.G., Hayes, S.F., Benach, J.L., Grunwald, E. and Davis, J.P. (1982) Science 216, 13171319.
[3] Barbour, A.G., Burgdorfer, W., Hayes, S.F., Peter, O. and Aeschlimann, A. (1983) Curr. Microbiol. 8, 123-126. [4] Sambri, V. and Lovett, M.A. (1990) Microbiologica 13, 79-83. [5] Iron Sub-committee (1978) Analyst 103, 391-396. [6] Hoepelman, I.M., Bezemer, W.A., Vandenbroucke-Grauls, C.M., Marx, J.J. and Verhoef, J. (1990) Infect. Immun. 58, 26-31. [7] Van Furth, R., Van Zwet, L. and Leijh, P.C.J. (1979) in Cellular Immunology (Weir, D.M., Ed.), 3rd Edn., pp. 32.1-32.19, Blackwell Scientific, Oxford. [8] Benach, J.L., Fleit, H.B., Habicht, G.S., Coleman, J.L., Bosler, E.M. and Lane, B.P. (1984) J. Infect. Dis. 150, 497-507. [9] Brock, J.H. (1986) Br. Med. J. 293, 518-519. [10] Jones, R.L., Peterson, C.M., Grady, R.W., Kumbaraci, T. and Cerami, A. (1977) Nature (London) 26, 63-69. [11] Repine, J.E., Fox, R.B., Berger, E.M. and Harada, R.N. (1981) Infect. Immun. 32, 407-410.
