N. Orsi, L. Seganti, and L. Sinibaldi, Atti del. Congresso Nazionale di Microbiologia, Fiuggi,. June 2-4 1978, in press) using conalbumin, which has a mechanism ...
Vol. 11, No. 5
JOURNAL OF CLINICAL MICROBIOLOGY, May 1980, p. 445-447 0095-1137/80/05-0445/03$02.00/0
Capacity of Staphylococci to Grow in the Presence of Ovotransferrin or CrCl3 as a Character of Potential Pathogenicity PIERA VALENTI,* ANDREA DE STASIO, LUCILLA SEGANTI, PAOLA MASTROMARINO, LAURA SINIBALDI, AND NICOLA ORSI Istituto di Microbiologia, Universita di Roma, Citta Universitaria, 00100 Rome, Italy
A total of 150 strains of staphylococci, clinically isolated, were tested for sensitivity to ovotransferrin (conalbumin). Among these, all the 50 coagulasepositive, mannitol-positive, and deoxyribonuclease-positive staphylococci appeared to be resistant to conalbumin, i.e., capable of growing in the presence of this transferrin. Among the other 100 strains, which were not classified as S. aureus, some appeared to be resistant and some were sensitive. The different behavior toward conalbumin is related to varying degrees of efficiency of the bacterial iron transport systems and, to test this, a simple method can be used, based on the addition of CrCb6 to the culture medium. The precipitation of iron produced by chromium salts has an effect on the growth of staphylococci similar to that produced by conalbumin and reveals the differences in the iron transport systems which occur in the genus Staphylococcus. Iron is an essential element for living things, by reason of its noticeable activity in electron transport reactions in biological systems (8). To procure and distribute this iron, which would be insoluble at physiological pH, all living beings have evolved iron transport systems based on iron-binding proteins and chelating agents (12). This happens also in bacteria, in which these siderophores (7) can have a different chemical structure and vary from species to species as it appears from studies on enterochelin of Escherichia coli and Salmonella typhimurium, schizochinine of Bacillus megaterium, phenolates of Bacillus cereus and Bacillus subtilis, and mycobactine of Mycobacterium smegmatis (4, 9). When bacteria behave as pathogens, they must acquire iron from the host and, therefore, compete with the host's transferrins in what has been defined as a "battle of chelating agents" (6). This aspect has been considered in experiments carried out with various types of transferrins and different bacterial species (1, 3, 16). With regard to the genus Staphylococcus in particular, it was observed by some authors (5, 10) that coagulase-positive staphylococci were resistant to serum transferrin, in contrast to coagulase-negative staphylococci which were sensitive. In addition, a preliminary investigation carried out by us (P. Valenti, A. De Stasio, N. Orsi, L. Seganti, and L. Sinibaldi, Atti del Congresso Nazionale di Microbiologia, Fiuggi, June 2-4 1978, in press) using conalbumin, which has a mechanism of action similar to the transferrins of mammals (1), showed that, among the
strains examined, all the coagulase-positive staphylococci were resistant to a certain concentration of conalbumin, whereas the coagulasenegative staphylococci were sensitive or resistant. In the present paper, data obtained in further experiments are reported, and a simple method is suggested for detecting the differences in the iron transport systems which occur in the genus Staphylococcus. MATERIALS AND METHODS Bacterial strains. A total of 150 strains of clinically isolated staphylococci was used. Among these, 50 were classified as Staphylococcus aureus, 50 were classified as Staphylococcus epidermidis, and 50 were classified as Staphylococcus saprophyticus, according to the classification of the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Staphylococci and Micrococci (11). Conalbumin. The freeze-dried preparations of conalbumin (kindly supplied by E. Antonini, Institute of Chemistry, Faculty of Medicine, University of Rome) were characterized by electrophoresis and iron-binding capacity. In starch gel electrophoresis, the protein gave a single band. Spectrophotometric titrations at 470 nm with iron citrate, at pH 9, in the presence of 0.1 M NaHCO3 gave endpoints corresponding to an iron-binding capacity of 1 + 0.05 microequivalents for 40 mg of protein. This value corresponds closely to the theoretical binding capacity of two iron atoms per molecule of protein of molecular weight 80,000. Conalbumin-sensitivity tests. The experiments were carried out with conalbuniin (10 mg/ml) dissolved in brain heart infusion (BHI) (BBL Microbiology Systems) with the addition of 50 mM NaHCO3. 445
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J. CLIN. MICROBIOL.
The iron content of the medium was 20 ug/g as determined with an atomic absorption spectrophotometer (Perkin-Elmer model 360). The inoculum consisted of about 106 bacterial cells. The growth, at 370C, was followed for 24 h by means of a Bonet-Maury-Jouan registering biophotometer. CrC13 sensitivity tests. The experiments were carried out in BHI medium, with the addition of 2% agar. The initial iron content of this agar was of 80 ,ug/g but, after treatment with ethylenediaminetetraacetate, it was reduced to 10 ,ug/g. To this medium, CrC13 was added to the final concentrations of 5 mM and 10 mM, both of which were able to produce a complete precipitation of the iron. This was demonstrated by the absence of iron in the supernatant after removal of the precipitate obtained by addition of CrC13 to BHI medium. The medium was poured in petri dishes in which the strains were tested by means of multiple needle inoculations, and the capacity to grow in the presence of CrC13 was detected after 24 h of incubation at 37°C. To verify whether or not the inhibition of the growth was due to the toxicity of Cr3+, controls were carried out by using BHI-agar with added chromium citrate at 5 mM and 10 mM.
RESULTS AND DISCUSSION An initial investigation of the sensitivity of 150 strains of staphylococci to conalbumin showed that all the 50 coagulase-positive and mannitol-positive strains examined were capable of growing in the presence of 10 mg of conalbumin per ml, whereas among the coagulase-negative strains, only a part was resistant (Fig. 1). With regard to other biochemical properties, such as the production of phosphatase and deoxyribonuclease, the distribution of the percentages of sensitive and resistant strains appears in Fig. 2. As to the 50 conalbumin-resistant strains of Staphylococcus aureus, it should be noted that they were all phosphatase positive and deoxyribonuclease positive. These results suggest that resistance to con100 '/
albumin can be considered an interesting parameter to evaluate, among the biological characteristics correlated to the potential pathogenicity of staphylococci. The presence of this character could be ascertained by demonstrating the capacity of a strain of Staphylococcus to grow in the presence of a certain concentration of conalbumin but, to carry out this test, it would be necessary to dispose of pure conalbumin or, at least, of a preparation of known biological activity. To overcome this difficulty, our research was directed to attempt a new test based not on the use of a transferrin, but on the addition to the culture medium of a certain concentration of CrC13. The choice of this substance is due to the fact that chromium salts precipitate iron contained in the culture medium and, in consequence of this, do not allow the growth of those bacteria with a lower degree of efficiency in the transport of iron. Research on this subject has already been carried out on E. coli (13-15), a species in which it was also possible to study the behavior of mutants specifically concerning the system of siderophores. The concentrations of CrCl3 we used (5 mM and 10 mM) were chosen after a series of tests carried out on the basis of the iron content of the culture medium employed, and the results obtained are reported in Table 1. It appears from this that a very high correlation can be demonstrated in staphylococci between the resistance to conalbumin and the capacity to grow in a culture medium with an appropriate amount of CrCl3 added. In our experimental conditions, this correlation was demonstrated with 10 mg of conalbumin per ml and a 5 mM concentration of CrC13, 100
S trains
%Str ains
80
80
60
60 40 40 20 20 0
Cog9ulase# Mannitol
#
0
CoagUlase
-
Coagulase
-
annitol
*
Mannitol
-
FIG. 1. Correlation between coagulase production and mannitol fermentation in staphylococci and their growth in the presence of ovotransferrin (10 mg/ml). c, Percent resistant strains; m_ percent sensitive strains.
-II ELI
DNAase
DNAa;.
Phoiphatase.
Phosphatase -
FIG. 2. Correlation between deoxyribonuclease and phosphatase production in staphylococci and their growth in the presence of ovotransferrin (10 mg/ ml). co, Percent resistant strains; _ percent sensitive strains.
SENSITIVITY OF STAPHYLOCOCCI TO OVOTRANSFERRIN
VOL. 11, 1980
TABLE 1. Frequency of strains resistant to conalbumin and CrCl3 No. resistant/no. tested Staphylococcus examined
Coagulase positive, mannitol positive Coagulase negative, mannitol positive Coagulase negative, mannitol negative
Conalr1 bumin (10 mg/ml) (5 mM)
CC3 (10 mM)
50/50
50/50
28/50
26/50
26/50
2/50
30/50
30/50
8/50
respectively, whereas with a 10 mM concentration of CrC13, the percentage of resistant staphylococci appeared to be lower, mainly in the case of coagulase-negative staphylococci. This fact indicates that among the strains of staphylococci there is a gradient of resistance to iron-chelating agents which is the expression of differences in the efficiency of the bacterial iron transport systems. Coagulase-positive staphylococci appeared to be, in general, capable of a higher resistance, but there are even some resistant strains among the coagulase-negative staphylococci. As to the importance of the presence of highly efficient iron transport systems for the pathogenicity of staphylococci, further research will be necessary, taking into account that it has been recently suggested that the action of transferrin can consist not only in a simple mechanism of iron withholding but can involve a more complex interaction between transferrin and bacterial surface (2). The results we obtained indicate that it is possible to use a simple culture medium containing CrCl3 to ascertain the capacity of a staphylococcus to resist the inhibiting action of transferrin and, on the basis of this test, to obtain additional information on the biological characteristics that in these bacteria may be related to pathogenicity.
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LITERATURE CITED 1. Antonini, E., N. Orsi, and P. Valenti. 1977. Effetto delle transfemne sulla patogenicita delle Enterobacteriaceae. G. Mal. Infet. Parassit. 29:481-489. 2. Arnold, R. R., M. F. Cole, and J. R. McGhee. 1977. A bactericidal effect for human lactoferrin. Science 197: 263-265. 3. Bullen, J. J., H. J. Rogers, and E. Griffiths. 1978. Role of iron in bacteria infection. Cur. Top. Microbiol. Immunol. 80:1-35. 4. Byers, B. R. 1974. Iron transport in gram-positive and acid fast bacilli, p. 83-105. In J. B. Neilands (ed.), Microbial iron metabolism. Academic Press Inc., New York. 5. Gladstone, G. P., and E. Walton. 1971. The effect of iron and haematin on the killing of staphylococci by rabbit polymorphs. Br. J. Exp. Pathol. 52:452-464. 6. Glynn, A. A. 1972. Bacterial factors inhibiting host defence mechanisms. Symp. Soc. Gen. Microbiol. 22:7581. 7. Lankford, C. E. 1973. Bacterial assimilation of iron. Crit. Rev. Microbiol. 2:273-331. 8. Neilands, J. B. 1974. Iron and its role in microbial physiology, p. 3-34. In J. B. Neilands (ed.), Microbial iron metabolism. Academic Press Inc., New York. 9. Rosemberg, H., and I. G. Young. 1974. Iron transport in the enteric bacteria, p. 67-82. In J. B. Neilands (ed.), Microbial iron metabolism. Academic Press Inc., New York. 10. Schade, A. L. 1963. Significance of serum iron for the growth, biological characteristics and metabolism of Staphylococcus aureus. Biochem. Z. 338:140-148. 11. Subcommittee on the Taxonomy of Staphylococci and Micrococci. 1975. Identification of Staphylococci. In Staphylococci and staphylococcal diseases. III International Symposium on Staphylococci and Staphylococcal Infections. Warszawa, September 8-14, p. 129. Gustav Fischer, Verlag, Stuttgart. 12. Sussman, M. 1974. Iron and infection, p. 649-679. In A. Jacobs and M. Worwood (ed.), Iron in biochemistry and medicine. Academic Press Inc., London. 13. Wang, C. C., and A. Newton. 1969. Iron transport in Escherichia coli: relationship between chromium sensitivity and high iron requirement in mutants of Escherichia coli. J. Bacteriol. 98:1135-1141. 14. Wang, C. C., and A. Newton. 1969. Iron transport in Escherichia coli: role of energy-dependent uptake and 2,3-dihydroxybenzoylserine. J. Bacteriol. 98:1142-1150. 15. Wang, C. C., and A. Newton. 1971. An additional step in the transport of iron defined by the ton B locus of Escherichia coli. J. Biol. Chem. 246:2147-2151. 16. Weiberg, E. D. 1974. Iron and susceptibility to infectious disease. Science 184:952-956.
