Zbl. Bakt. 276, 481-486 (1992) © Gustav Fischer Verlag, StuttgartlNew York
Siderophore Production of Klebsiella Species Isolated from Different Sources RAINER PODSCHUN, ARNO FISCHER, and UWE ULLMANN Institute of Medical Microbiology, University of Kiel, D-2300 Kiel
Received October 8, 1991 . Accepted November 5, 1991
Summary A total of 481 Klebsiella pneumoniae and K. oxytoca strains isolated from different sources was examined for siderophore production. Screening for siderophore secretion by chrome azurol S agar revealed that 475 strains (98.8%) produced siderophores. The isolates were further investigated for synthesis of enterochelin and aerobactin by means of specific bioassays. Almost all Klebsiella strains (99.4%) excreted enterochelin. Aerobactin production, however, was rarely observed among K. pneumoniae (6%) and K. oxytoca (4%) isolates. The incidence of aerobaction-positive strains was similar in clinical, fecal, and environmental isolates. These results suggest that the aerobactin system does not represent a major mechanism of iron supply in Klebsiella spp.
Zusammenfassung 481 Klebsiella pneumoniae- und K. oxytoca-Isolate verschiedener Herkunft wurden hinsichtlich der Bildung von Siderophoren untersucht. Durch Anziichtung auf Chrom Azurol SAgar konnte bei 475 Stammen (98,8%) eine Siderophorproduktion festgestellt werden. Die Isolate wurden mittels spezifischer Bioteste auf die Bildung von Enterochelin und Aerobaktin hin gepriift. Nahezu aIle Stamme (99,4%) exprimierten Enterochelin. 1m Gegensatz dazu wurde eine Exkretion von Aerobaktin bei K. pneumoniae (6%) und K. oxytoca (4%) sehr selten beobachtet. Die Haufigkeit Aerobaktin-positiver Stamme war bei Isolaten aus klinischem Material, den Faces sowie aus der Umwelt ahnlich gering. Diese Ergebnisse deuten darauf hin, daB Aerobaktin bei Klebsiellen keine Schliisselstellung in der Eisenversorgung dieser Bakterien einnimmt.
Introduction The element iron is essential for the growth of bacteria. Since body fluids contain little free iron, bacterial pathogens have evolved a variety of mechanisms of iron acquisition. Many bacteria excrete low-molecular-weight iron-chelating compounds, known as siderophores. In enteric bacteria, two types of high-affinity iron chela tors have been described: the catechol type siderophore enterochelin (enterobactin) and the
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hydroxamate type aerobactin. Most clinical isolates of Escherichia coli and Salmonella produce enterochelin (3, 6, 14) whereas aerobactin is less common (7). While the role of enterochelin is uncertain, aerobactin is considered to be an important virulence factor. In Shigella sp., Klebsiella pneumoniae, and invasive strains of E. coli the contribution of aerobactin production to virulence has been demonstrated (4, 8, 9, 21, 22). Epidemiologically, aerobactin production has been found more frequently in clinical isolates of E. coli than among strains isolated from the feces of healthy individuals or from the environment (1, 11, 20). Although K. pneumoniae has been shown to produce both enterochelin and aerobactin (2, 8, 12, 15,23), little information exists on the distribution of siderophore production among Klebsiella spp. The aim of the present study was to evaluate the incidence of enterochelin and aerobactin expression among different Klebsiella species isolated from various sources. We investigated K. pneumoniae and K. oxytoca strains isolated from clinical material, from the feces of healthy individuals, and from the environment. Materials and Methods
Bacteria. A total of 481 Klebsiella strains that have been described previously (13) were examined: 128 K. pneumoniae and 53 K. oxytoca isolates were from human clinical specimens, 109 K. pneumoniae and 54 K. oxytoca strains were isolated from the feces of healthy individuals (food handlers), 106 K. pneumoniae and 31 K. oxytoca strains were sewage isolates. All isolates had been frozen at -80°C immediately after isolation. After thawing, strains were routinely cultivated on China blue lactose agar (Merck). E. coli strains H1939, H188?, and K311 were kindly provided by K. Hantke, University of Tiibingen, Germany. H1939 is an FhuK, FhuB- mutant of Hl728 (FepA +, Fiu-, Cir-, aroB) (5) that is defective in the uptake of hydroxamates and catechols other than enterochelin. The strain was used as an enterochelin indicator strain, as it is unable to synthesize enterochelin and thus will grown in iron-limited media only if supplied with this siderophore by another strain. H1887 (FepA-, Fiu-, Cir-, aroB), which carries the plasmid pEN41 (ColV-, Aer-, lut+), is defective in aerobactin and enterochelin biosynthesis and lacks receptors for catechols. The strain will grow under iron-limited conditions if provided with aerobactin but not enterochelin. Strain K311 (pColV-K311) is a producer of aerobactin and served as positive control in the aerobactin test. Chemicals. All chemicals and reagents were of analytical grade. Chrome azurol S (CAS) was obtained from Fluka Chemie, hexadecyltrimethylammonium bromide (HDTMA) was purchased from E. Merck. 1,4-piperazinediethanesulphonic acid (Pipes) and 2,2'-dipyridyl was procured from Sigma Chemical Company. All glassware was immersed overnight in 5% (voUvol) Extran (Merck) and rinsed in double-distilled water. After further overnight soaking in 0.01 % (wtlvol) EDTA, the glassware was rinsed in 1% HCl and six times in double-distilled water. Siderophore screening. Strains were screened for production of siderophores with a universal chemical assay described by Schwyn and Neilands (17). Briefly, this medium contains a blue coloured indicator complex composed of chrome azurol S (CAS), iron(III), and hexadecyltrimethylammonium bromide (HDTMA). Siderophores release the iron, causing the dye complex to change colour. CAS agar (pH 6.8) consists of 0.1 roM CAS, 0.2 mM HDTMA, 0.01 mM FeCI3 , MM9 salts, 100 roM Pipes, 15 g agar, and 6 g NaOH per litre, and was supplemented with 0.3% casamino acids, 0.2% glucose, and 2 mg thiamine· HC!. Isolates and reference strains were spotted on CAS agar and incubated for 20 h at 37°C. Siderophore-producing strains showed large yellow halos around the colonies. Each isolate was tested twice.
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Bioassay for aerobactin and enterochelin production. The type of siderophore produced was determined by a cross-feeding assay described by Hantke (5). E. coli H1887 was used as indicator strain for aerobactin production, and strain H1939 for enterochelin. Indicator strains were grown overnight in nutrient broth (Oxoid) at 37°C. Cells were harvested by centrifugation and diluted 1: 10 in physiological saline to 107_10 8 cells/ml. 100111 of bacterial suspension were plated on nutrient agar (Oxoid) to which 2,2'-dipyridyl (200 11M) had been added to make it iron-limited. Cultures of the strains to be tested and positive controls were spotted on the agar plates and incubated for 24 h. A halo of growth of the particular indicator strain around the test strain indicated production of aerobactin or enterochelin. Each isolate was tested twice. Results The strains were screened for siderophore production with a universal chemical assay. By means of CAS agar, 475 of the 481 Klebsiella strains investigated (98.8%) were found to secrete siderophores (Table 1). Siderophore-negative strains were observed only among sewage isolates of K. pneumoniae (4%) and among clinical isolates of K. oxytoca (4%). Table 1. Incidence of siderophore production (detected by CAS agar) and secretion of enterochelin and aerobactin among Klebsiella isolates from different sources Strains
K. pneumoniae
sewage (n = 106) fecal (n = 109) clinical (n = 128)
K.oxytoca
sewage (n = 31) fecal (n = 54) clinical (n = 53)
Total (n = 481)
Number of strains (percentage) secreting Siderophores Enterochelin Aerobactin 102 (96) 109 (100) 128 (100)
105 (99) 109 (100) 128 (100)
7 (6.6) 6 (5.5) 7 (5.5)
31 (100) 54 (100) 51 (96)
31 (100) 54 (100) 51 (96)
0(0) 3 (5.6) 3 (5.7)
475 (98.8)
478 (99.4)
26 (5.4)
Determination of enterochelin excretion revealed that almost all strains (99.4%) produced this type of siderophore. Only one K. pneumoniae sewage strain and two clinical isolates of K. oxytoca did not synthesize enterochelin. In three sewage strains of K. pneumoniae that were siderophore-negative on CAS agar, enterochelin secretion could be detected. In contrast to the high incidence of enterochelin synthesis in Klebsiella strains, aerobactin production was rarely observed (5%). The frequencies of aerobactin-positive isolates were similar in K. pneumoniae (5.8%) and K. oxytoca (4.3%). Except for sewage isolates of K. oxytoca, among which no aerobactin-producing strain at all was observed, no differences in the incidence of aerobactin production were detected between environmental, fecal, and clinical strains.
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Discussion Chrome azurol S (CAS) agar proved to be a useful tool for screening large numbers of strains with respect to siderophore production. When spotted on agar plates, up to 25 isolates could be tested per plate. In most cases, siderophore-positive strains showed a large halo of colour change around the colonies. In five strains, a rather small zone of colour change was observed. It could, however, be identified as positive without any difficulty in comparison with the negative control. Three out of 479 isolates (0.6%) that were positive in the bioassays were observed to be negative on CAS agar. Taken together, CAS agar was highly sensitive, and the results were easy to interpret. Almost all Klebsiella isolates examined (98.8%) were found to secrete siderophores. By means of specific bioassays, the strains were further tested for production of enterochelin and aerobactin. Enterochelin is produced by enteric bacteria of the genera Escherichia, Salmonella, Klebsiella, and some species of Shigella in vitro and in vivo (12, 14, 16, 19). Since almost all clinical isolates of Escherichia coli and Salmonella synthesize this siderophore type (3, 6), enterochelin is regarded as a 'basal' siderqphore of enteric bacteria. Enteochelin also seems to be the main siderophore produced by Klebsiella spp.: Regardless of their origin, all but three K. pneumoniae and K. oxytoca isolates we observed were enterochelin-positive. This is in agreement with the findings of Williams et al. (24) who found all of 17 clinical Klebsiella isolates to excrete enterochelin. Another type of iron chelator, the hydroxamate siderophore aerobactin, is produced by some members of the family Enterobacteriaceae. Aerobactin synthesis has been correlated with the virulence of K. pneumoniae and invasive strains of E. coli (8, 21, 22). While enterochelin is secreted by most strains of enteric bacteria, aerobactin is less common. The incidence of aerobactin expression seems to depend on the species: Martinez et al. (7) divided enteric bacteria into a group with high frequency of aerobactin production (> 40%) consisting of Escherichia, Shigella, and Enterobacter, and a group with low incidence of aerobactin-positive strains « 20%). The latter group included the genera Salmonella and Klebsiella. Twelve of 67 clinical Klebsiella isolates examined (18%) were found to synthesize aerobactin. Similarly, we observed a low frequency of aerobactin expression among Klebsiella strains. Only 6% of the isolates we investigated secreted aerobactin, and no difference in the incidence of aerobactinpositive strains was found between the species K. pneumoniae and K. oxytoca. In E. coli, the incidence of aerobactin expression has been found to depend upon the source of isolation. Blood isolates show the highest frequency of aerobactin expression (11, 20). Generally, clinical E. coli isolates synthesize aerobactin more frequently than fecal or environmental strains (1, 10, 11, 20). A low incidence of aerobactin-positive strains (6%) has been found in E. coli from drinking water and effluent (20). This percentage is comparable to that found by us among environmental strains of K. pneumoniae. In contrast to E. coli, however, Klebsiella isolates from feces and from clinical material synthesized aerobactin as rarely as did environmental isolates, suggesting that aerobactin is not a principal mechanism of iron acquisition in Klebsiella spp. In this respect, Klebsiella resembles Salmonella spp. which usually do not produce aerobactin. Epidemiologically, our findings do not indicate that this siderophore contributes significantly to the virulence of Klebsiella. The aerobactin concept also has other shortcomings: the higher affinity of enterochelin for iron makes it difficult to explain why aerobactin should have a selective advantage in infection. In spite of the post-
Siderophore Production of Klebsiella Species
485
ulated importance of aerobactin expression for extracellular growth, a substantial fraction of aerobactin-negative E. coli clinical isolates can grown in the host tissue (18), suggesting that other iron uptake mechanisms are involved. The role of enterochelin in infection is unclear, as so far no Ene, Aer+ E. coli strain has been found and investigated. In conclusion, our data do not provide evidence that aerobactin is an important factor in the virulence of Klebsiella. Among K. pneumoniae and K. oxytoca isolates, aerobactin synthesis is a rare event, and it certainly does not represent a major mechanism of iron acquisition in these species.
References
1. Carbonetti, N. H., S. Boonchai, S. H. Parry, V. Viiisiinen-Rhen, T. K. Korhonen, and P. H. Williams: Aerobactin-mediated iron uptake by Escherichia coli isolates from human extraintestinal infections. Infect. Immun. 51 (1986) 966-968
2. Gibson, F. and D. I. Magrath: The isolation and characterisation of a hydroxamic acid (aerobactin) formed by Aerobacter aerogenes. Biochim. Biophys. Acta 192 (1969) 175-184
3. Griffiths, E. and J. Humphreys: Isolation of enterochelin from the peritoneal washings of guinea pigs lethally infected with Escherichia coli. Infect. Immun. 28 (1980) 286-289 4. Griffiths, E., P. Stevenson, T.L. Hale, and S. B. Formal: Synthesis of aerobactin and a 76,000-Dalton iron-regulated outer membrane protein by Escherichia coli K12 - Shigella flexneri hybrids and by enteroinvasive strains of Escherichia coli. Infect. Immun. 49 (1985) 67-71
5. Hantke, K.: Dihydroxybenzoylserine - a siderophore for E. coli. FEMS Microbiol. Lett. 67(1990)5-8
6. Kochan, I., J. Wasynckaz, and A. McCabe: Effect of injected iron and siderophores on infection in normal and immune mice. Infect. Immun. 22 (1978) 560-567
7. Martinez, ]. L., J. L. Cercenado, F. Baquero, J. C. Perez Diaz, and A. Delgado-Iribarren: Incidence of aerobactin production in gram-negative hospital isolates. FEMS Microbiol. Lett. 43 (1987) 351-353
8. Nassif, X. and P. ]. Sansonetti: Correlation of the virulence of Klebsiella pneumoniae
Kl and K2 with the presence of a plasmid encoding aerobactin. Infect. Immun. 54 (1986) 603-608 9. Nassif, x., M. C. Mazert,]. Mounier, and P.]. Sansonetti: Evaluation with an iuc: TnlO mutant of the role of aerobactin production in the virulence of Shigella flexneri. Infect. Immun. 55 (1987) 1963-1969 10. 0rskov, I., C. Svanborg-Eden, and F. 0rskov: Aerobactin production of serotyped Escherichia coli from urinary tract infections. Med. Microbiol. Immunol. 177 (1988) 9-14 11. Opal, S. M., A. S. Cross, P. Gemski, and L. W. Lyhte: Aerobactin and a-hemolysin as virulence determinants in Escherichia coli isolated from human blood, urine, and stool. J. Infect. Dis. 161 (1990) 794-796 12. Perry, R. D. and c.L. San Clemente: Siderophore synthesis in Klebsiella pneumoniae and Shigella sonnei during iron deficiency. J. Bact. 140 (1979) 1129-1132 13. Podschun, R.: Phenotypic properties of Klebsiella pneumoniae and K. oxytoca isolated from different sources. Zbl. Hyg. 189 (1990) 527-535 14. Pollack, J. R. and J. B. Neilands: Enterobactin, an iron transport compound from Salmonella typhimurium. Biochem. Biophys. Res. Commun. 38 (1970) 989-992 15. Reissbrodt, R. and W. Rabsch: Further differentiation of Enterobacteriaceae by means of siderophore-pattern analysis. Zbl. Bakt. Hyg. A 268 (1988) 306-317
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16. Rogers, H. J., c. Synge, B. Kimber, and P. M. Bayley: Production of enterochelin by Escherichia coli 0111. Biochim. Biophys. Acta 497 (1977) 548-557 17. Schwyn, B. andj. B. Neilands: Universal chemical assay for the detection and determination of siderophores. Analyt. Biochem. 160 (1987) 47-56
18. Smith, H. W. and M.B. Huggins: The association of the 018 K1 and H7 antigens and the ColV plasmid of a strain of Escherichia coli with its virulence and immunogenicity.
J. Gen. Microbiol.
121 (1980) 387-400
19. Struelens, M. j., G. Mondal, M. Roberts, and P. H. Williams: Role of bacterial and host factors in the pathogenesis of Shigella bacteremia. Eur. J. Clin. Microbiol. Infect. Dis. 9 (1990) 337-344
20. Stuart, S. j., K. T. Greenwood, and R. K. j. Luke: Iron-suppressible production of hydroxamate by Escherichia coli isolates. Infect. Immun. 36 (1982) 870-875 21. Williams, P. H.: Novel iron uptake system specified by ColV plasmids: an important component in the virulence of invasive strains of Escherichia coli. Infect. Immun. 26 (1979) 925-932
22. Williams, P. H. and P. j. Warner: ColV plasmid-mediated colicin V-independent iron uptake system of invasive strains of Escherichia coli. Infect. Immun. 29 (1980) 411-416 23. Williams, P., M. R. W. Brown, and P. A. Lambert: Effect of iron deprivation on the production of siderophores and outer mem.bratle proteins in Klebsiella aerogenes. J. Gen. Microbiol. 130 (1984) 2357-2365
24. Williams, P., H. Chart, E. Griffiths, and P. Stevenson: Expression of high affinity iron uptake systems by clinical isolates of Klebsiella. FEMS Microbiol. Lett. 44 (1987) 407-412
Dr. R. Podschun, Institut fUr Medizinische Mikrobiologie und Virologie, Universitat Kiel, Brunswiker Str. 4, D-2300 Kiel, Germany
Siderophore Production of Klebsiella Species Isolated from Different Sources RAINER PODSCHUN, ARNO FISCHER, and UWE ULLMANN Institute of Medical Microbiology, University of Kiel, D-2300 Kiel
Received October 8, 1991 . Accepted November 5, 1991
Summary A total of 481 Klebsiella pneumoniae and K. oxytoca strains isolated from different sources was examined for siderophore production. Screening for siderophore secretion by chrome azurol S agar revealed that 475 strains (98.8%) produced siderophores. The isolates were further investigated for synthesis of enterochelin and aerobactin by means of specific bioassays. Almost all Klebsiella strains (99.4%) excreted enterochelin. Aerobactin production, however, was rarely observed among K. pneumoniae (6%) and K. oxytoca (4%) isolates. The incidence of aerobaction-positive strains was similar in clinical, fecal, and environmental isolates. These results suggest that the aerobactin system does not represent a major mechanism of iron supply in Klebsiella spp.
Zusammenfassung 481 Klebsiella pneumoniae- und K. oxytoca-Isolate verschiedener Herkunft wurden hinsichtlich der Bildung von Siderophoren untersucht. Durch Anziichtung auf Chrom Azurol SAgar konnte bei 475 Stammen (98,8%) eine Siderophorproduktion festgestellt werden. Die Isolate wurden mittels spezifischer Bioteste auf die Bildung von Enterochelin und Aerobaktin hin gepriift. Nahezu aIle Stamme (99,4%) exprimierten Enterochelin. 1m Gegensatz dazu wurde eine Exkretion von Aerobaktin bei K. pneumoniae (6%) und K. oxytoca (4%) sehr selten beobachtet. Die Haufigkeit Aerobaktin-positiver Stamme war bei Isolaten aus klinischem Material, den Faces sowie aus der Umwelt ahnlich gering. Diese Ergebnisse deuten darauf hin, daB Aerobaktin bei Klebsiellen keine Schliisselstellung in der Eisenversorgung dieser Bakterien einnimmt.
Introduction The element iron is essential for the growth of bacteria. Since body fluids contain little free iron, bacterial pathogens have evolved a variety of mechanisms of iron acquisition. Many bacteria excrete low-molecular-weight iron-chelating compounds, known as siderophores. In enteric bacteria, two types of high-affinity iron chela tors have been described: the catechol type siderophore enterochelin (enterobactin) and the
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hydroxamate type aerobactin. Most clinical isolates of Escherichia coli and Salmonella produce enterochelin (3, 6, 14) whereas aerobactin is less common (7). While the role of enterochelin is uncertain, aerobactin is considered to be an important virulence factor. In Shigella sp., Klebsiella pneumoniae, and invasive strains of E. coli the contribution of aerobactin production to virulence has been demonstrated (4, 8, 9, 21, 22). Epidemiologically, aerobactin production has been found more frequently in clinical isolates of E. coli than among strains isolated from the feces of healthy individuals or from the environment (1, 11, 20). Although K. pneumoniae has been shown to produce both enterochelin and aerobactin (2, 8, 12, 15,23), little information exists on the distribution of siderophore production among Klebsiella spp. The aim of the present study was to evaluate the incidence of enterochelin and aerobactin expression among different Klebsiella species isolated from various sources. We investigated K. pneumoniae and K. oxytoca strains isolated from clinical material, from the feces of healthy individuals, and from the environment. Materials and Methods
Bacteria. A total of 481 Klebsiella strains that have been described previously (13) were examined: 128 K. pneumoniae and 53 K. oxytoca isolates were from human clinical specimens, 109 K. pneumoniae and 54 K. oxytoca strains were isolated from the feces of healthy individuals (food handlers), 106 K. pneumoniae and 31 K. oxytoca strains were sewage isolates. All isolates had been frozen at -80°C immediately after isolation. After thawing, strains were routinely cultivated on China blue lactose agar (Merck). E. coli strains H1939, H188?, and K311 were kindly provided by K. Hantke, University of Tiibingen, Germany. H1939 is an FhuK, FhuB- mutant of Hl728 (FepA +, Fiu-, Cir-, aroB) (5) that is defective in the uptake of hydroxamates and catechols other than enterochelin. The strain was used as an enterochelin indicator strain, as it is unable to synthesize enterochelin and thus will grown in iron-limited media only if supplied with this siderophore by another strain. H1887 (FepA-, Fiu-, Cir-, aroB), which carries the plasmid pEN41 (ColV-, Aer-, lut+), is defective in aerobactin and enterochelin biosynthesis and lacks receptors for catechols. The strain will grow under iron-limited conditions if provided with aerobactin but not enterochelin. Strain K311 (pColV-K311) is a producer of aerobactin and served as positive control in the aerobactin test. Chemicals. All chemicals and reagents were of analytical grade. Chrome azurol S (CAS) was obtained from Fluka Chemie, hexadecyltrimethylammonium bromide (HDTMA) was purchased from E. Merck. 1,4-piperazinediethanesulphonic acid (Pipes) and 2,2'-dipyridyl was procured from Sigma Chemical Company. All glassware was immersed overnight in 5% (voUvol) Extran (Merck) and rinsed in double-distilled water. After further overnight soaking in 0.01 % (wtlvol) EDTA, the glassware was rinsed in 1% HCl and six times in double-distilled water. Siderophore screening. Strains were screened for production of siderophores with a universal chemical assay described by Schwyn and Neilands (17). Briefly, this medium contains a blue coloured indicator complex composed of chrome azurol S (CAS), iron(III), and hexadecyltrimethylammonium bromide (HDTMA). Siderophores release the iron, causing the dye complex to change colour. CAS agar (pH 6.8) consists of 0.1 roM CAS, 0.2 mM HDTMA, 0.01 mM FeCI3 , MM9 salts, 100 roM Pipes, 15 g agar, and 6 g NaOH per litre, and was supplemented with 0.3% casamino acids, 0.2% glucose, and 2 mg thiamine· HC!. Isolates and reference strains were spotted on CAS agar and incubated for 20 h at 37°C. Siderophore-producing strains showed large yellow halos around the colonies. Each isolate was tested twice.
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483
Bioassay for aerobactin and enterochelin production. The type of siderophore produced was determined by a cross-feeding assay described by Hantke (5). E. coli H1887 was used as indicator strain for aerobactin production, and strain H1939 for enterochelin. Indicator strains were grown overnight in nutrient broth (Oxoid) at 37°C. Cells were harvested by centrifugation and diluted 1: 10 in physiological saline to 107_10 8 cells/ml. 100111 of bacterial suspension were plated on nutrient agar (Oxoid) to which 2,2'-dipyridyl (200 11M) had been added to make it iron-limited. Cultures of the strains to be tested and positive controls were spotted on the agar plates and incubated for 24 h. A halo of growth of the particular indicator strain around the test strain indicated production of aerobactin or enterochelin. Each isolate was tested twice. Results The strains were screened for siderophore production with a universal chemical assay. By means of CAS agar, 475 of the 481 Klebsiella strains investigated (98.8%) were found to secrete siderophores (Table 1). Siderophore-negative strains were observed only among sewage isolates of K. pneumoniae (4%) and among clinical isolates of K. oxytoca (4%). Table 1. Incidence of siderophore production (detected by CAS agar) and secretion of enterochelin and aerobactin among Klebsiella isolates from different sources Strains
K. pneumoniae
sewage (n = 106) fecal (n = 109) clinical (n = 128)
K.oxytoca
sewage (n = 31) fecal (n = 54) clinical (n = 53)
Total (n = 481)
Number of strains (percentage) secreting Siderophores Enterochelin Aerobactin 102 (96) 109 (100) 128 (100)
105 (99) 109 (100) 128 (100)
7 (6.6) 6 (5.5) 7 (5.5)
31 (100) 54 (100) 51 (96)
31 (100) 54 (100) 51 (96)
0(0) 3 (5.6) 3 (5.7)
475 (98.8)
478 (99.4)
26 (5.4)
Determination of enterochelin excretion revealed that almost all strains (99.4%) produced this type of siderophore. Only one K. pneumoniae sewage strain and two clinical isolates of K. oxytoca did not synthesize enterochelin. In three sewage strains of K. pneumoniae that were siderophore-negative on CAS agar, enterochelin secretion could be detected. In contrast to the high incidence of enterochelin synthesis in Klebsiella strains, aerobactin production was rarely observed (5%). The frequencies of aerobactin-positive isolates were similar in K. pneumoniae (5.8%) and K. oxytoca (4.3%). Except for sewage isolates of K. oxytoca, among which no aerobactin-producing strain at all was observed, no differences in the incidence of aerobactin production were detected between environmental, fecal, and clinical strains.
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Discussion Chrome azurol S (CAS) agar proved to be a useful tool for screening large numbers of strains with respect to siderophore production. When spotted on agar plates, up to 25 isolates could be tested per plate. In most cases, siderophore-positive strains showed a large halo of colour change around the colonies. In five strains, a rather small zone of colour change was observed. It could, however, be identified as positive without any difficulty in comparison with the negative control. Three out of 479 isolates (0.6%) that were positive in the bioassays were observed to be negative on CAS agar. Taken together, CAS agar was highly sensitive, and the results were easy to interpret. Almost all Klebsiella isolates examined (98.8%) were found to secrete siderophores. By means of specific bioassays, the strains were further tested for production of enterochelin and aerobactin. Enterochelin is produced by enteric bacteria of the genera Escherichia, Salmonella, Klebsiella, and some species of Shigella in vitro and in vivo (12, 14, 16, 19). Since almost all clinical isolates of Escherichia coli and Salmonella synthesize this siderophore type (3, 6), enterochelin is regarded as a 'basal' siderqphore of enteric bacteria. Enteochelin also seems to be the main siderophore produced by Klebsiella spp.: Regardless of their origin, all but three K. pneumoniae and K. oxytoca isolates we observed were enterochelin-positive. This is in agreement with the findings of Williams et al. (24) who found all of 17 clinical Klebsiella isolates to excrete enterochelin. Another type of iron chelator, the hydroxamate siderophore aerobactin, is produced by some members of the family Enterobacteriaceae. Aerobactin synthesis has been correlated with the virulence of K. pneumoniae and invasive strains of E. coli (8, 21, 22). While enterochelin is secreted by most strains of enteric bacteria, aerobactin is less common. The incidence of aerobactin expression seems to depend on the species: Martinez et al. (7) divided enteric bacteria into a group with high frequency of aerobactin production (> 40%) consisting of Escherichia, Shigella, and Enterobacter, and a group with low incidence of aerobactin-positive strains « 20%). The latter group included the genera Salmonella and Klebsiella. Twelve of 67 clinical Klebsiella isolates examined (18%) were found to synthesize aerobactin. Similarly, we observed a low frequency of aerobactin expression among Klebsiella strains. Only 6% of the isolates we investigated secreted aerobactin, and no difference in the incidence of aerobactinpositive strains was found between the species K. pneumoniae and K. oxytoca. In E. coli, the incidence of aerobactin expression has been found to depend upon the source of isolation. Blood isolates show the highest frequency of aerobactin expression (11, 20). Generally, clinical E. coli isolates synthesize aerobactin more frequently than fecal or environmental strains (1, 10, 11, 20). A low incidence of aerobactin-positive strains (6%) has been found in E. coli from drinking water and effluent (20). This percentage is comparable to that found by us among environmental strains of K. pneumoniae. In contrast to E. coli, however, Klebsiella isolates from feces and from clinical material synthesized aerobactin as rarely as did environmental isolates, suggesting that aerobactin is not a principal mechanism of iron acquisition in Klebsiella spp. In this respect, Klebsiella resembles Salmonella spp. which usually do not produce aerobactin. Epidemiologically, our findings do not indicate that this siderophore contributes significantly to the virulence of Klebsiella. The aerobactin concept also has other shortcomings: the higher affinity of enterochelin for iron makes it difficult to explain why aerobactin should have a selective advantage in infection. In spite of the post-
Siderophore Production of Klebsiella Species
485
ulated importance of aerobactin expression for extracellular growth, a substantial fraction of aerobactin-negative E. coli clinical isolates can grown in the host tissue (18), suggesting that other iron uptake mechanisms are involved. The role of enterochelin in infection is unclear, as so far no Ene, Aer+ E. coli strain has been found and investigated. In conclusion, our data do not provide evidence that aerobactin is an important factor in the virulence of Klebsiella. Among K. pneumoniae and K. oxytoca isolates, aerobactin synthesis is a rare event, and it certainly does not represent a major mechanism of iron acquisition in these species.
References
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Dr. R. Podschun, Institut fUr Medizinische Mikrobiologie und Virologie, Universitat Kiel, Brunswiker Str. 4, D-2300 Kiel, Germany
