Acinetobacter baumannii - Journal of Bacteriology - American

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JOURNAL OF BACTERIOLOGY, Dec. 1992, p. 7670-7679 Vol. 174, No. 23 0021-9193/92/237670-10$02.00/0 Copyright ) 1992, American Society for Microbiology Characterization of a High-Affinity Iron Transport System in Acinetobacter baumannii JOSE R. ECHENIQUE,1 HECTOR ARIENTI,1 MARCELO E. TOLMASKY,2t RON R. READ,3 ROBERTO J. STANELONI,2 JORGE H. CROSA,3 AND LUIS A. AC11S1t* Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba,1 and Instituto de Investigaciones Bioquimicas "Fundacion Campomar, " Buenos Aires 2 Argentina, and Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201-3o983 Received 6 April 1992/Accepted 30 September 1992 Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the SalmoneUla typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extraceliular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(M) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 ,uM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes. Acinetobacter baumannii, formerly known asAcinetobac- ter calcoaceticus subsp. anitratus (15), belongs to a bacterial species that is widely distributed throughout the environ- ment (6). This microorganism can be isolated from the skin (3, 43) and respiratory tract (59) of healthy ambulatory adults as well as from hospital personnel and equipment (36, 39, 46, 67). Lately, numerous outbreaks of nosocomial infections caused by A. baumannii have been reported (7-10, 16, 42, 47, 56, 57, 73), which are of particular concern because of the widespread and increasing antibiotic resistance of the isolated strains (17, 30, 32, 33, 40, 41, 48). Nevertheless, most infections have occurred in patients compromised by either antibiotic therapy, respiratory instrumentation and manipulations, dialysis, or surgery (18, 19, 39, 70). Thus, it was proposed that reduced host defenses and interaction with other bacterial species, rather than the expression of specific bacterial virulence factors (49), are the factors responsible for opportunistic infections caused by Acineto- bacter species. Acinetobacter species are able to survive under restricted nutrient conditions, such as those imposed by the host. Iron is one of the essential bacterial nutrients that is tightly controlled by the host, through its chelation by the high- affinity binding glycoproteins transferrin (TF) and lactoferrin (23). Thus, bacteria have developed efficient iron uptake mechanisms that allow them to scavenge iron from these host proteins, either by direct interaction with the iron- protein complexes (34) or by the synthesis and secretion of * Corresponding author. t Present address: Department of Microbiology and Immunology L220, School of Medicine, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201-3098. high-affinity extracellular siderophore compounds (23). It was recently reported that clinical isolates of A. baumannii produce 2,3-dihydroxybenzoic acid (DHBA) when grown in a chemically defined medium and can utilize Fe-DHBA complexes to grow in the presence of the iron chelator 2,2'-bipyridyl (DIP) (66). In this report, we describe the isolation and preliminary characterization of an iron-regulated catechol compound capable of promoting the uptake of 55Fe(III) as well as the growth of A. baumannii in the presence of human TF. MATERIALS AND METHODS Bacterial strains and plasmids. The clinical strain A. bau- mannii 8399 was isolated during a nosocomial outbreak of respiratory tract infections (39). Vibrio anguillarum 531A (69) was used as source of anguibactin, a catechol-type siderophore different from enterobactin (1). The Escherichia coli LG1522 mutant, which is deficient in aerobactin synthe- sis but carries the receptor for this siderophore, was used as the indicator strain to detect aerobactin production (72). Salmonella typhimurium mutants enb-1, which can use only enterobactin, and enb-7, which can use enterobactin as well as DHBA (55) to grow under iron-deficient conditions, were used as indicator strains to detect the production of entero- bactin and DHBA, respectively. Plasmids pCP111 (20) and pCP410 (54) were used as enterobactin probes. Plasmid pABN5 (11) was used as the aerobactin probe. Growth conditions. Trypticase soy agar and broth, L agar and broth, M9 minimal medium (22), and Fe-CDM chemi- cally defined medium (66) were used to culture the bacterial strains. The iron chelators human TF, nitrilotriacetic acid (NTA), DIP, and ethylenediaminedi-(o-hydroxyphenyl)ace- tic acid (EDDA; Sigma Chemical Co., St. Louis, Mo.) were 7670 Downloaded from https://journals.asm.org/journal/jb on 16 December 2021 by 191.240.119.209.

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Characterization of a high-affinity iron transport system in Acinetobacter baumanniiJOURNAL OF BACTERIOLOGY, Dec. 1992, p. 7670-7679 Vol. 174, No. 23 0021-9193/92/237670-10$02.00/0 Copyright ) 1992, American Society for Microbiology
Characterization of a High-Affinity Iron Transport System in Acinetobacter baumannii
JOSE R. ECHENIQUE,1 HECTOR ARIENTI,1 MARCELO E. TOLMASKY,2t RON R. READ,3 ROBERTO J. STANELONI,2 JORGE H. CROSA,3 AND LUIS A. AC11S1t*
Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba,1 and Instituto de Investigaciones Bioquimicas "Fundacion Campomar, " Buenos Aires 2
Argentina, and Department ofMicrobiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201-3o983
Received 6 April 1992/Accepted 30 September 1992
Analysis of a clinical isolate ofAcinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the SalmoneUla typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extraceliular catechol was confirmed by its ability to stimulate energy-dependent uptake of55Fe(M) as well as to promote the growth ofA. baumannii bacterial cells under iron-deficient conditions imposed by 60 ,uM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate ofA. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.
Acinetobacter baumannii, formerly known asAcinetobac- ter calcoaceticus subsp. anitratus (15), belongs to a bacterial species that is widely distributed throughout the environ- ment (6). This microorganism can be isolated from the skin (3, 43) and respiratory tract (59) of healthy ambulatory adults as well as from hospital personnel and equipment (36, 39, 46, 67).
Lately, numerous outbreaks of nosocomial infections caused by A. baumannii have been reported (7-10, 16, 42, 47, 56, 57, 73), which are of particular concern because of the widespread and increasing antibiotic resistance of the isolated strains (17, 30, 32, 33, 40, 41, 48). Nevertheless, most infections have occurred in patients compromised by either antibiotic therapy, respiratory instrumentation and manipulations, dialysis, or surgery (18, 19, 39, 70). Thus, it was proposed that reduced host defenses and interaction with other bacterial species, rather than the expression of specific bacterial virulence factors (49), are the factors responsible for opportunistic infections caused by Acineto- bacter species. Acinetobacter species are able to survive under restricted
nutrient conditions, such as those imposed by the host. Iron is one of the essential bacterial nutrients that is tightly controlled by the host, through its chelation by the high- affinity binding glycoproteins transferrin (TF) and lactoferrin (23). Thus, bacteria have developed efficient iron uptake mechanisms that allow them to scavenge iron from these host proteins, either by direct interaction with the iron- protein complexes (34) or by the synthesis and secretion of
* Corresponding author. t Present address: Department of Microbiology and Immunology
L220, School of Medicine, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201-3098.
high-affinity extracellular siderophore compounds (23). It was recently reported that clinical isolates ofA. baumannii produce 2,3-dihydroxybenzoic acid (DHBA) when grown in a chemically defined medium and can utilize Fe-DHBA complexes to grow in the presence of the iron chelator 2,2'-bipyridyl (DIP) (66).
In this report, we describe the isolation and preliminary characterization of an iron-regulated catechol compound capable of promoting the uptake of 55Fe(III) as well as the growth ofA. baumannii in the presence of human TF.
MATERIALS AND METHODS
Bacterial strains and plasmids. The clinical strain A. bau- mannii 8399 was isolated during a nosocomial outbreak of respiratory tract infections (39). Vibrio anguillarum 531A (69) was used as source of anguibactin, a catechol-type siderophore different from enterobactin (1). The Escherichia coli LG1522 mutant, which is deficient in aerobactin synthe- sis but carries the receptor for this siderophore, was used as the indicator strain to detect aerobactin production (72). Salmonella typhimurium mutants enb-1, which can use only enterobactin, and enb-7, which can use enterobactin as well as DHBA (55) to grow under iron-deficient conditions, were used as indicator strains to detect the production of entero- bactin and DHBA, respectively. Plasmids pCP111 (20) and pCP410 (54) were used as enterobactin probes. Plasmid pABN5 (11) was used as the aerobactin probe. Growth conditions. Trypticase soy agar and broth, L agar
and broth, M9 minimal medium (22), and Fe-CDM chemi- cally defined medium (66) were used to culture the bacterial strains. The iron chelators human TF, nitrilotriacetic acid (NTA), DIP, and ethylenediaminedi-(o-hydroxyphenyl)ace- tic acid (EDDA; Sigma Chemical Co., St. Louis, Mo.) were
7670
IRON ACQUISITION IN A. BAUMANNII 7671
added to the media to achieve iron-limiting conditions. TF was prepared as a 1 mM stock solution in 100 mM Tris-HCl (pH 7.5)-0.15 M NaCl-50 mM NaHCO3. The MICs for the iron chelators were determined in either liquid or solid media containing increasing concentrations of each iron-chelating compound. Iron-proficient conditions were obtained by add- ing 50 p.M FeCl3 in 0.5 M HCl. The inocula used for growth and "Fe(III) uptake studies as well as for siderophore purification were prepared by using cells iron starved by culturing in M9 or Fe-CDM medium containing 50 p.M EDDA or DIP.
Cloacin sensitivity and TF binding assays. Cloacin was
prepared from Enterobacter cloacae DF13 as previously described (71). Purified cloacin was spotted onto L agar
plates containing a lawn of the bacterial strain to be tested and increasing concentrations of EDDA. Bacterial growth was recorded after overnight incubation at 37°C. E. coli LG1315 (72) was used as a positive control. The ability of bacteria to bind TF directly was assayed by
the method of Schryvers and Morris (61), using a horserad- ish peroxidase (HRP) conjugate (Pierce, Rockford, Ill.). A clinical isolate of Haemophilus influenzae was used as a
positive control. Siderophore production. The production of extracellular
compounds with siderophore activity was investigated by using the method of Schwyn and Neilands (62). The pres-
ence of phenolic or hydroxamate extracellular compounds was detected in culture supernatants with the Arnow phe- nolic acid assay (4) or the Csaky hydroxylamine/hydroxamic acid assay (27), respectively. Catechol siderophores were
extracted with ethyl acetate from iron-deficient culture su-
pernatants adjusted to pH 1.5 with HCl (58). The ethyl acetate samples were dried and dissolved in either ethanol or
phosphate-buffered saline. Hydroxamate siderophores were
extracted with chloroform-phenol from culture supernatants adjusted to pH 7.0 as described by Simpson and Oliver (64).
Siderophore bioassays. Siderophore activity was deter- mined in liquid or solid bioassays by testing the ability of cell-free culture supernatants to promote growth of bacterial strains under iron-limiting conditions (2). Aerobactin synthe- sis was determined as previously described (26), using E. coli LG1522 as the indicator strain. The presence of entero- bactin and DHBA was examined by plate assays, using the S. typhimurium enb-1 and enb-7 mutants, as previously described (44). The purified siderophores enterobactin, amonabactin, and pyochelin and pyoverdin were generously provided by J. B. Neilands, B. R. Byers, and C. Cox, respectively.
Radioactive iron uptake. A protocol similar to that de- scribed for V. anguillarum (2) was used. Briefly,A. bauman- nii 8399 was grown at 37°C for 12 h in 30 ml of M9 containing 50 p.M EDDA. The cells were washed and suspended in the same volume of M9 medium containing only Casamino Acids and 50 p.M EDDA. After incubation for 1 h, the cells were collected, washed, resuspended in M9 medium without Casamino Acids and containing 100 pM NTA, and further incubated for 1 h at 37°C. Cell concentration was adjusted to an A6. of 0.4 with the same medium. The 15Fe(III)-sidero- phore complex was prepared by incubating the purified Acinetobacter siderophore with 55FeC13 (specific activity, 62.10 mCi/mg; NEN Research Products, DuPont Co., Bos- ton, Mass.) at room temperature for 10 min in M9 salts. The final concentration of 55FeC13 in the uptake reaction was 0.13 p.Ci/ml. Since the structure and composition of the Acineto- bacter siderophore are not known, its concentration was
determined as DHBA equivalents by the Arnow test, using 1
mM DHBA as the standard solution. The final concentration of the Acinetobacter siderophore in the uptake mixtures, as DHBA equivalents, was 12 p,M. The uptake assays were initiated by the addition of 55FeCl3 or 55Fe(III)-siderophore to the cell suspensions. Samples of 1 ml were removed after 5 and 10 min of incubation at 37°C and immediately filtered through 0.45-p,m-pore-size HA filters (Millipore Corp., San Francisco, Calif.) in a vacuum-drawn manifold. The filters were washed with 5 ml of 0.1 M sodium citrate. The amount of 5"Fe(III) retained on the filters was measured by liquid scintillation counting.
Cells suspensions were preincubated for 30 min at 37°C with either 20 mM potassium cyanate (KCN) or 1 mM carbonyl m-chlorophenylhydrazone (CCCP5) (Sigma) to ex- amine whether the A. baumannii 8399 5Fe(III) uptake system requires cell energy.
Siderophore purification. The extracellular siderophore(s) secreted by A. baumannii 8399 was isolated from iron- deficient culture supernatants by adsorption chromatogra- phy onto XAD-7 resin (1). Cells were grown for 16 h at 37°C in minimal medium. A 4-liter volume of culture supernatant, adjusted to neutral pH, was applied to an XAD-7 column (2.5 by 17 cm). The column was washed with 2 volumes of deionized water, and the adsorbed material was eluted with pure methanol. Chromatographic fractions containing cate- chol and siderophore compounds were identified by using the Arnow and Chrome azurol S (CAS) reactions, respec- tively. The peak column fractions containing the iron-bind- ing activity were concentrated by evaporation at reduced pressure and kept at 4°C protected from the light. TLC and paper chromatography. Thin-layer chromatogra-
phy (TLC) was performed on 0.2-mm precoated Silica Gel 60 F-254 TLC sheets (E. Merk, Elmsford, N.Y.), using one of the following solvent systems: chloroform-methanol (2:1), butanol-acetic acid-water (60:15:25), benzene-acetic acid- water (125:72:3), and butanol-pyridine-water (14:3:3). Paper chromatography was performed on 3MM chromatography paper (Whatman Ltd., Maidstone, England), using one of the following solvent systems: chloroform-methanol (2:1), chloroform-methanol-water (10:20:3), and butanol-acetic ac- id-water (60:15:25). The chromatograms were examined under UV light, and iron-binding compounds were detected by spraying either the CAS reagent or 0.1 M FeCl3 in 0.1 M HCl. Catechol compounds were detected by spraying the reagents of the Arnow test. Mass spectrometry. Fast atom bombardment spectra were
obtained on a Kratos MS5OTC mass spectrometer at the Department of Agricultural and Life Sciences, Oregon State University. Samples were analyzed in either 3-nitrobenzoic acid or dithioerythritol-dithiothreitol matrixes. DNA techniques. Total DNA from the A. baumannii 8399
was obtained by the method described by Meade et al. (45). Plasmid DNA was isolated from E. coli HB101 (13) by the method of Birnboim and Doly (12) and further purified by centrifugation in CsCl-ethidium bromide density gradients (60). Southern blot hybridizations were performed as de- scribed previously (68). DNA probes were obtained by the oligolabeling method described by Feinberg and Vogelstein (31), using [a-32P]dATP. Electrophoresis of DNA restriction fragments was performed in horizontal slab gels as previ- ously described (25). Restriction enzymes were used as described by the supplier (Bethesda Research Laboratories, Gaithersburg, Md.). DNA restriction fragments were iso- lated from agarose gels by using Geneclean (Bio 101, La Jolla, Calif.).
Electrophoretic analysis of membrane proteins. Overnight
VOL. 174, 1992
7672 ECHENIQUE ET AL.
TABLE 1. Cell growth and siderophore production by A. baumannii 8399 under iron-deficient and
iron-proficient conditions
Compound Concn Cell Colorimetric reaction in medium (PM) growta CASb Arnowc Csakyd
TF 30 + + + NDe DIP 120 + + + ND EDDA 800 + + + - NTA 800 + + + ND FeCl3 50 + - ND
a Determined spectrophotometrically at 600 nm. Optical densities were >1.0 after overnight incubation. b A color change from blue to pink-orange was considered a positive
reaction. c See reference 4. d See reference 27.e ND, not determined.
bacterial cultures were washed by centrifugation, resus- pended in 10 mM Tris-HCl (pH 8.0) containing 0.3% NaCl, and sonically disrupted at 4°C. Total cell envelopes were prepared as described previously (24), and the outer mem- brane fractions were obtained by differential solubilization of the cell envelopes, using 1.5% Sarkosyl. Total and outer membrane proteins (100 ,ug) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), using 12.5% separating gels (2). After electrophore- sis (30 mA for 7 h), the gels were stained with Coomassie blue and then destained with 5% acetic acid. Protein con- centrations were determined as described by Bradford (14).
RESULTS Bacterial growth under iron-limiting conditions. The initial
experiments showed that the clinical isolate A. baumannii 8399, obtained during a nosocomial outbreak of lower respi- ratory tract infections, was able to grow in M9 minimal medium containing either 50 p,M EDDA or 10 ,uM TF, suggesting that this strain was iron uptake proficient. Deter- mination of the MICs revealed that this isolate can grow in the presence of 30 ,uM TF as well as in M9 medium containing up to 800 p,M EDDA or NTA or up to 120 ,uM DIP (Table 1). These results indicated that this bacterial strain acquires iron from chelated medium, either through direct interaction with ferrated TF or through an efficient iron uptake system that may involve an extracellular sidero- phore. HRP-TF binding assays showed that iron-starved A. bau-
mannii 8399 cells did not bind the labeled human TF (Fig. 1). Conversely, the H. influenzae strain used as a positive control bound TF under the same experimental conditions. Therefore, it seems that the direct interaction between A. baumannii cells and human HRP-labeled TF is not required to scavenge the iron from the metal-protein complexes. The production of extracellular siderophore(s) was then
investigated by using the CAS reagent as described by Schwyn and Neilands (62). The appearance of orange halos around the bacterial colonies on CAS-agar plates revealed the production and secretion of an iron chelator compound (data not shown). Furthermore, the addition of iron-limited, but not iron-rich, culture supernatants to the CAS reagent produced a color change of the reagent from blue to orange (Table 1). These results strongly suggested thatA. bauman- nii 8399 excreted a siderophore compound into the iron- limited culture medium.
1
2
3
4 FIG. 1. Binding of HRP-labeled human TF by H. influenzae (1)
and A. baumannii 8399 cells grown in M9 minimal medium contain- ing either 50 ,uM FeCl3 (2), 50 ,uM EDDA (3), or 100 F.M EDDA (4).
Characterization of the A. baumannii siderophore. The chemical nature of the siderophore was determined by using the Arnow (4) and Csaky (27) colorimetric reactions. Cate- chol compounds were detected in iron-deficient culture supernatants with the Arnow test, while no reaction was observed with the Csaky test (Table 1), indicating that no hydroxamate compounds were present in the medium. The uninoculated culture medium gave negative results with both colorimetric tests. To confirm these results, A. baumannii 8399 iron-chelated culture supernatants were treated with ethyl acetate or phenol-chloroform, to extract and concen- trate either catechol or hydroxamate siderophores, respec- tively. The ethyl acetate extracts were positive for cate- chols, while the phenol-chloroform extracts remained hydroxamate negative. We also investigated at the biological and genetic levels the ability of A. baumannii 8399 to produce aerobactin. Biologically, the presence of this hy- droxamate siderophore was ruled out since utilization bio- assays capable of detecting it were negative. Furthermore, the cloacin sensitivity test, which can detect the presence of the aerobactin receptor in bacterial membranes, showed that A. baumannii 8399 was cloacin resistant, suggesting the absence of this outer membrane receptor. Genetically, no homology was detected between A. baumannii 8399 total DNA and the aerobactin genes probe pABN5 by DNA hybridization under stringent conditions (data not shown). Thus, only the production of a catechol siderophore(s) could be detected in culture supernatants. This extracellular cate- chol(s) was detected in the culture medium of cells grown at between 30 and 42°C, with the highest levels after overnight culture at 37°C (Fig. 2a). Figure 2b shows thatA. baumannii 8399 cells grew properly under iron-deficient conditions, although they exhibited an increased generation time and a reduced growth yield compared with bacteria cultured in iron-proficient conditions (Fig. 2c). The secretion of cate- chol(s) paralleled the bacterial growth in iron-deficient M9 minimal medium, reaching the maximum after 24 h of incubation (Fig. 2b), whereas the amount of this com- pound(s) in iron-rich medium was below the detectable levels of the Arnow test (Fig. 2c).
Siderophore utilization bioassays showed that iron-defi- cient culture supernatants of this bacterium were able to reverse the EDDA inhibitory effect and enhance the growth of the S. typhimurium enb-7 mutant (Table 2), an iron uptake-deficient mutant that can utilize DHBA as a precur-
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FIG. 2. (a) Synthesis and secretion of catechol compounds in culture supernatants ofA. baumannii 8399 grown at 32, 37, or 42°C in M9 minimal medium containing different amounts of EDDA; (b and c) growth (optical density at 600 nm [OD 600 nm]) and catechol synthesis (optical density at 510 nm) by A. baumannui 8399 in the presence of either 100 ,uM EDDA (b) or 50 FM FeCl3 (c). Cells were cultured 24 h in M9 minimal medium at 37C with constant shaking. Catechol compounds were detected by the method of Arnow (4).
TABLE 2. Siderophore utilization bioassays
S. tphimwium growth halos (mm)b Material tested0
enb-1 enb-7
FeCl3 14 15 Enterobactin 12 13 V. anguillarum 531A 0 10 A. baumannu 8399 0 7
a Samples of sterile culture supernatants from either V. anguillanan 531A or A. baumannii 8399 were added to filter disks and placed on top of solid medium. FeCl3 and enterobactin were used as positive controls.
b M9 minimal medium agar plates containing 50 ,M EDDA were seeded with S. typhimwium enb-1 orenb-7 iron uptake mutant cells. Diameters of the growth halos were recorded after 16 h of incubation at 37C.
sor to produce enterobactin (55). A similar effect was de- tected in iron-deficient culture supernatants of V. anguil- larum 531A, a bacterial strain that also produces DHBA together with anguibactin, a catechol siderophore unrelated to enterobactin (2, 69). Conversely, the V. anguillarum 531A andA. baumannii 8399 culture supernatants were not able to promote the growth of the S. typhimurium enb-1 mutant (Table 2), an iron uptake mutant that requires the addition of enterobactin to grow under iron limitation (55). Thus, DHBA but not enterobactin is secreted into the growth medium by A. baumannii 8399. This observation was further supported by the fact that the enterobactin genes could not be detected in the genome of this bacterium by DNA hybridization analysis (data not shown), using as labeled probes plasmids pCP111 and pCP410, which harbor the enterobactin genes fepC, fepB, and entF (pCP111) and entA, entC, !entG, entB, and entE (pCP410) (20, 54).
Isolation of theA. baumannii siderophore. The facts thatA. baumannii 8399 is able to grow in the presence of high concentrations of TF and secretes an iron-regulated com- pound(s) reacting with the CAS reagent suggest that this bacterium produces, besides DHBA, an extracellular sidero- phore that is not related to enterobactin. To prove this hypothesis, iron-deficient culture supernatants were sub- jected to adsorption chromatography on XAD-7 resin. Cul- ture supernatants adjusted to pH 7.0, to avoid the coadsorp- tion of low-molecular-weight organic acids (DHBA among them) (1), were applied to a chromatographic column. A peak of catechol compound(s), with very low siderophore activity as determined with the CAS reagent, was eluted when the column was washed with 2 volumes of deionized water (Fig. 3). Elution of the XAD-7 column with pure methanol yielded a well-defined catechol peak that coeluted with a very significant peak of siderophore activity (Fig. 3). The same elution profiles were obtained with use of either M9 or Fe-CDM minimal medium, although the latter gave clearer results due to the utilization of arginine, aspartate, proline, and glutamic acid instead of Casamino Acids, thus avoiding the pigments that are normally present in the commercial Casamino Acids preparations and that are also retained in the XAD-7 column (data not shown).
Analysis by TLC of the pooled peak fractions eluted with pure methanol revealed a single component by all visualiza- tion methods. This iron-binding catechol component mi- grated very poorly on the silica plates, showing R values different from those of DHBA and enterobactin (Table 3). Similar results were obtained by paper chromatography, although two components with R1 of 0.06 and 0.44 were detected by using the butanol-acetic acid-water solvent system.
0
0
FRACTIONS
FIG. 3. XAD-7 column chromatography of A. baumannii 8399 culture supernatant. Deionized water (dH20) and pure methanol (METOH) elution steps are indicated by the arrows. Elution of catechol(s) was monitored with the Arnow reaction (510 nm) (4). Siderophore activity of chromatographic fractions was determined with the CAS reagent (630 nm) (62). The decrease in absorbance of the CAS reagent is inversely related to the siderophore activity present in each sample. Therefore, the activity of each fraction is represented as the inverse of the decrease ofA630 with respect to the absorbance of the blank reaction (minimal medium plus CAS reagent). The siderophore activity could not be determined on all of the catechol-containing fractions eluted with pure methanol because of interference by this organic solvent.
UV-visible light (VIS) spectrum analysis also showed that the purified catechol is different from DHBA (Fig. 4A). Similar spectra were obtained whether the purified catechol was dissolved in methanol or aqueous solution (Fig. 4A). The UV-VIS spectrum of this catechol compound was
further compared with the spectra described for other bac- terial catechol siderophores, using the experimental condi- tions described for analysis of the latter. The spectrum of the Acinetobacter catechol in aqueous solution at pH 7.5 shown in Fig. 4A is different from those reported for anguibactin, the siderophore isolated from V. anguillarum 775, which exhibits absorption maxima at 214, 260, 315, and 410 nm (1), and pseudobactin 589A, an iron-chelating agent isolated from Pseudomonas putida 589, which has a maximum ab- sorption peak at 403 nm (53). In addition, the spectra of enterobactin and the Acinetobacter catechol were different when both iron-chelating compounds were dissolved in methanol (data not shown). Figure 4B shows that the spec- trum of theAcinetobacter catechol dissolved in HCl-ethanol is different from those described for agrobactin and vibrio-
bactin, the siderophores isolated from Agrobacterium tume- faciens (50) and Vibrio cholerae (35), respectively. In addi- tion, the isolated catechol did not show the broad peak at 316 nm and the sharp peak at 252 nm described for agrobactin solubilized in ethanol (50) (data not shown). The absorption spectrum of the Acinetobacter catechol at pH 6.5 in 0.1 M morpholinopropanesulfonic acid (MOPS) exhibited an
absorbance maximum at 324 nm and a shoulder at 260 nm (Fig. 4B), values that are different from those reported for chrysobactin, a catechol-type siderophore isolated from iron-deficient culture supernatants of the plant pathogen Erwinia chrysanthemi (52). This UV-VIS spectral analysis also showed that the electronic absorption spectra of amon- abactin, the catechol siderophore found in Aeromonas hy- drophila (5), and the Acinetobacter catechol are different (Fig. 4C). The siderophores pyochelin and pyoverdin, iso- lated from iron-deficient Pseudomonas culture supematants (21, 29), showed absorption spectra different from those of the Acinetobacter catechol when examined at pH 5.0 (Fig. 4D). Figure 4D also shows that the Acinetobacter catechol does not exhibit at pH 5.0 the peak at 380 nm and the shoulder at 336 nm described for azotobactin D, a catechol siderophore produced by Azotobacter vinelandii D (28). Mass spectral analysis of the XAD-7 column fraction
containing this catechol compound gave a prominent ion at mlz 391.3, corresponding to [MH]+ in the cationic detection mode. The mass of this ion, which may represent the Acinetobacter catechol that possesses the siderophore activ- ity, is different from the values reported for the siderophores agrobactin, amonabactin, anguibactin, azotobactin, chryso- bactin, enterobactin, pseudobactin, pyochelin, pyoverdin, and vibriobactin, which were used in the UV-VIS analysis.
In conclusion, we have shown the existence of a catechol component in the culture supernatants ofA. baumannii 8399 which possesses iron-binding properties and appears to be different from DHBA and several other bacterial catechol- type siderophores.
Siderophore activity of the purified catechol. The sidero- phore activity of the purified component was tested by its ability to promote the growth of A. baumannii 8399 in the presence of increasing concentrations of TF. Figure 5 shows that the addition of this purified compound to M9 medium containing 60 ,uM TF stimulated the bacterial growth by a
factor of 5, and no effect was observed in the presence of 90 ,uM TF. Conversely, when DHBA was added to M9 medium in amounts equivalent to those of the purified catechol compound, as measured by the Arnow test, insignificant
TABLE 3. Rf values of the catechol component(s) purified by XAD-7 column chromatography from A. baumannu 8399 culture supernatants
Rf Support Solvent system A. baumnnii
8399 catechol DHBA Enterobactin
a ND, not determined.
020033 o 0 0.
Wavelength (nm) Wavelength (nm)
1.2 11.2
0.4 < 0.6
FIG. 4. WV-VIS absorption spectra of the purifiedA. baumannii 8399 catechol, DHBA, and some bacterial catechol sideropho'res. (A4) Spectra ofDHBA( )a'ndAcinetobacter catechol dissolved in either 0.05 M Tris-HCI-0.2M Na2SO4 (pH 7.5) ( )or methanol (O )v (B)spectra of the Acinetobacter catechol dissolved in either 10% 0.1 N HCI-90% ethanol (--)or 0.1 M MOPS (pH 6.5) ( ;(C) spectra of amonabactin (---)and theAcinetobacter catechol ( )dissolved in 75%o ethanol; (D) spectra of the Acinetobacter
catechol ( ,pyoverdin (-),and pyochelin (0-0) at pH 5.0. The spectra were recorded on a B'eckman' DU-40 spectrophotometer.
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0. 11
9. 20
TRANSFERRIN (i.M)
FIG. 5. Siderophore activity of the purified A. baumannii 8399 catechol. Bacterial growth was assayed in the presence of different concentrations of TF either in plain M9 medium (-) or in the presence ofA. baumannii 8399 catechol (1) or DHBA (U1). OD 600 nm, optical density at 600 nm.
increases in bacterial growth were detected at both 60 and 90 ,M TF. The involvement of this catechol in iron uptake was
confirmed by its ability to promote iron accumulation in iron-starved A. baumannii 8399 cells. Figure 6 shows that the uptake of 55Fe(III) was significantly enhanced by the addition of this compound to the uptake mixtures. The rate of this siderophore-mediated transport of 55Fe(III) is fast, with the maximum intracellular accumulation occurring within 5 min and remaining constant even after 20 min of incubation under the experimental conditions described in Materials and Methods. The energy dependence of this uptake process was determined by using the inhibitors CCCP and KCN. The addition of 1 mM CCCP to the uptake mixtures inhibited the uptake of 55Fe(III) in the presence of the purified catechol, while no effect was detected when 20 mM KCN was added, indicating that the uptake of 55Fe(III) in A. baumannii is coupled to proton movement across the membranes and not to the respiratory chain (63). These results strongly suggest that the catechol compound
isolated from A. baumannii 8399 culture supernatants is
0
20 -
10 -
0-
MINUTES
FIG. 6. "Fe(III) uptake by nongrowingA. baumannii 8399 cells under iron-limiting conditions. The intracellular accumulation of "FeCl3 was determined after the cells were incubated at 37°C for 5 and 10 min in the presence of either carrier-free 55FeC13 (-) or 55Fe(III)-A. baumannii 8399 siderophore (5,, , / ). The energy dependence of the uptake process was examined by adding either 20 mM KCN (U) or 1 mM CCCP ( , ).
134
120
87 -82
77 \76
70 68
FIG. 7. SDS-PAGE of total membranes (lanes 1 and 2) and outer membrane proteins (lanes 3 and 4) fromA. baumannii 8399 grown in M9 minimal medium containing either 50 p,M FeCl3 (lanes 2 and 4) or 100 p,M EDDA (lanes 1 and 3). Molecular weight marker proteins (lane M) were phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), trypsin inhibitor (20,100), and a-lac- talbumin (14,200).
indeed an extracellular siderophore, capable of scavenging iron from high-affinity iron chelators such as human TF and bringing it into the bacterial cells across the membranes in an energy-dependent mode.
Effect of iron on the expression of A. baumannii 8399 membrane proteins. The effect of iron on the synthesis of membrane proteins was investigated by SDS-PAGE. Several membrane proteins are induced under iron-limiting condi- tions (Fig. 7, lanes 1 and 2). Treatment of the total mem- branes with 1.5% Sarkosyl revealed that most of these iron-regulated proteins are located in the outer membrane of strain 8399 (lanes 1 and 3). However, two of them, of 120 and 134 kDa, appear to be localized in the inner membrane, since they are present in the total membrane preparation (lane 1) but absent in the detergent-treated sample (lane 3). Con- versely, the latter sample contains at least seven iron- regulated proteins, with molecular masses ranging from 68 to 87 kDa (lane 3). The fact that these inner and outer mem- brane proteins are expressed only under iron limitation suggests that some of them may be involved in the recogni- tion and transport process for the iron-siderophore com- plexes.
DISCUSSION
A. baumannii is an opportunistic human pathogen that presents a potential risk of severe infections for patients who are compromised or suffer from polymicrobial infections. Thus, the simple presence of this bacterium is not sufficient for establishment of an infection, which is thus related to the clinical status of the colonized host. Furthermore, the nature of the factors affecting the virulence of this bacterium is not fully understood.
Analysis of experimental infections in mice (49) showed that the slime ofA. baumannii is a virulence factor that acts by impairing the functions and cell structure of mice phago- cytes. The concurrent intraperitoneal injection of slime- producing Acinetobacter strains or isolated slime enhances the virulence of E. coli, Serratia marcescens, and P. aerug- inosa strains with respect to single bacterial infections. However, this study showed that the virulence of A. bau-
J. BACTERIOL.
IRON ACQUISITION IN A. BAUMANNII 7677
mannii is very low, and no differences in 50% lethal dose were found between the slime-producing and non-slime- producing strains. More recently, Smith et al. (66) reported that a clinical
isolate of A. baumannii was able to grow in a chemically defined medium containing DIP. Chemical and chromato- graphic analysis of ethyl acetate extracts of culture superna- tants from that isolate revealed only the presence of DHBA, while bioassays showed that the organism could utilize Fe-DHBA complexes to grow under iron-restricted condi- tions. These findings suggested to the authors that A. bau- mannii expresses an iron uptake system involving DHBA as a putative extracellular siderophore. Other reports have also indicated that DHBA may act as a siderophore (37, 38, 51), although its activity appears to be somewhat disputable under the iron-restricted conditions described in the human host. Consequently, we initiated a detailed analysis of the iron proficiency phenotype of the A. baumannii 8399 strain isolated during a nosocomial outbreak of lower tract respi- ratory infections. The free iron concentration of the culture medium affected the growth of bacteria as well as the synthesis of membrane proteins and extracellular products. However, this strain grew well under the iron-limiting con- ditions imposed by different iron chelators added in high concentrations to chemically defined media. This iron scav- enging activity appears not to require a direct contact between the bacterial cells and the iron-chelating human glycoprotein, as is the case with H. influenzae, Neisseria meningitidis, and N. gonorrhoeae (34).
Colorimetric and siderophore utilization tests, supported by DNA hybridization experiments, revealed that the iron uptake proficiency of A. baumannii 8399 is related to the synthesis and excretion of an iron-regulated catechol sidero- phore and not to a hydroxamate iron-chelating agent. The siderophore utilization bioassays, the chromatographic be- havior, and the UV-VIS and mass spectral properties con- firmed these results. In addition, they indicated that the isolated catechol siderophore is different from agrobactin, amonabactin, anguibactin, azotobactin, chrysobactin, enter- obactin, pseudobactin, pyochelin, pyoverdin, and vibriobac- tin. However, the possibility that the Acinetobacter sidero- phore is either similar or identical to other catechol siderophores not included in our study will be clarified after the elucidation of its chemical structure. The actual siderophore activity of the purifiedA. bauman-
nii 8399 catechol was confirmed by utilization bioassays, since the addition of this compound to minimal medium containing 60 ,uM TF facilitated significantly the growth of A. baumannii 8399. Such growth facilitation was not ob- served when an equivalent amount of DHBA was added to the chemically defined culture medium containing the same TF concentration. These results also indicate that this cate- chol is a high-affinity siderophore, since it is able to scavenge iron from strong chelating molecules such as human TF. The role of this extracellular component in the iron uptake process was demonstrated by its ability to promote the intracellular accumulation of 55Fe(III). The fact that this uptake process is sensitive to CCCP and not to KCN indicates that it requires energy coupled to a transmembrane proton gradient rather than to the electron transport chain (63). Although it was shown that A. baumannii secretes and
utilizes DHBA as a siderophore (66), the fact that DHBA was added in that study as a preformed iron complex should be taken into account. The experimental approach described in that publication does not demonstrate that DHBA can
acquire iron from TF when the cells are cultured in the presence of this protein. Indeed, our results indicate that DHBA cannot promote the growth ofA. baumannii 8399 in minimal medium containing 60 ,uM TF, supporting the general concept that DHBA is a weak iron chelator that can provide iron only under mild restricted conditions. It has been reported that DHBA can stimulate E. coli growth on nutrient plates containing DIP or iron-deficient M9 minimal medium only if it is used as a precursor for the biosynthesis of enterobactin (37, 38). In the case ofA. vinelandii, DHBA was shown to act as an effective iron-solubilizing agent, but when free iron becomes less abundant, this bacterium com- bines the production of DHBA with production of the higher-affinity siderophores azotobactin and azotochelin (51). Thus, it is possible that A. baumannii 8399 can utilize
DHBA as a siderophore to grow in culture media containing moderately low levels of free iron, but it must produce a siderophore with higher affinity for iron and its cognate transport system to grow under more stringent iron-deficient conditions, such as those found in the human host. Our preliminary research in this work for such a high-
affinity transport system led to the identification of two inner membrane proteins and of at least seven outer membrane proteins that are expressed only when A. baumannii 8399 is grown under iron-limiting conditions. Some of these outer membrane proteins may correspond to those already identi- fied by Coomassie blue staining (66) or immunoblotting with serum from septicemic patients (65), although no iron- regulated inner membrane proteins have previously been detected in Acinetobacter species. Furthermore, some of these membrane proteins may play an important role as components of the cell membrane receptors that recognize and internalize the Fe-siderophore complexes, as was de- scribed for other bacterial iron uptake systems (23). The expression of this siderophore-mediated iron uptake
system could be another important factor in the pathogenesis of Acinetobacter infections, since such systems were dem- onstrated to be important virulence factors in the establish- ment of bacterial infections (23).
ACKNOWLEDGMENTS This work was supported by grants from Consejo Nacional de
Investigaciones Cientificas y Tecnicas (CONICET) of Argentina to L.A.A., M.E.T., and R.J.S. and from Consejo de Investigaciones Cientificas y Tecnologicas de la Provincia de Cordoba (Argentina) to L.A.A. and by Public Health Service grant AI-19018 from the National Institutes of Health to J.H.C. R.R.R. was supported by a Medical Research Council of Canada fellowship. M.E.T., R.J.S., and L.A.A. are Career Investigator members of CONICET. We are thankful to L. M. Crosa for valuable help in performing
the aerobactin utilization bioassays.
REFERENCES 1. Actis, L. A., W. Fish, J. H. Crosa, K. Kellerman, S. R.
Ellenberger, F. M. Hauser, and J. Sanders-Loehr. 1986. Char- acterization of anguibactin, a novel siderophore from Vibrio anguilarum 775(pJM1). J. Bacteriol. 167:57-65.
2. Actis, L. A., S. A. Potter, and J. H. Crosa. 1985. Iron-regulated outer membrane protein OM2 of Vibrio anguillarum is encoded by the virulence plasmid pJM1. J. Bacteriol. 161:736-742.
3. Al-Khoja, M. S., and J. H. Darrell. 1979. The skin as the source of Acinetobacter and Moraxella species occurring in blood cultures. J. Clin. Pathol. 32:497-499.
4. Arnow, L. E. 1937. Colorimetric determination of the compo- nents of 3,4-dihydroxyphenylalanine-tyrosine mixtures. J. Biol. Chem. 118:531-537.
5. Barghouthi, S., R. Young, M. 0. J. Olson, J. E. L. Arceneaux,
VOL. 174, 1992
L. W. Clem, and B. R. Byers. 1989. Amonabactin, a novel tryptophan- or phenylalanine-containing phenolate siderophore inAeromonas hydrophila. J. Bacteriol. 171:1811-1816.
6. Baumann, P. 1968. Isolation of Acinetobacter from soil and water. J. Bacteriol. 96:39-42.
7. Beck-Sague, C. M., W. R. Jaris, J. H. Brook, D. H. Culver, A. Potts, E. Gay, B. W. Shotts, B. Hill, R. L. Anderson, and M. P. Weinstein. 1990. Epidemic bacteremia due to Acinetobacter baumannii in five intensive care units. Am. J. Epidemiol. 132:723-733.
8. Bergogne-Berezin, E., and M. L. Joly-Guillou. 1985. An under- estimated nosocomial pathogen, Acinetobacter calcoaceticus. J. Antimicrob. Chemother. 16:535-538.
9. Bergogne-Berezin, E., and M. L. Joly-Guillou. 1991. Hospital infection with Acinetobacter spp.: an increasing problem. J. Hosp. Infect. 18:250-255.
10. Bergogne-Berezin, E., M. L. Joly-Guillou, and J. F. Vieu. 1987. Epidemiology of nosocomial infections due to Acinetobacter calcoaceticus. J. Hosp. Infect. 10:105-113.
11. Binderif, A., and J. B. Neilands. 1983. Cloning of the aerobactin- mediated iron assimilation system of plasmid ColV. J. Bacteriol. 153:1111-1113.
12. Birnboim, H., and J. Doly. 1979. A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523.
13. Boyer, H. W., and D. Roulland-Dussoix. 1969. A complementa- tion analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41:459-472.
14. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72:249-254.
15. Bouvet, P. J. M., and P. A. D. Grimont. 1986. Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Ac- inetobacterjohnsonii sp. nov., andAcinetobacterjunii sp. nov. and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter Iwoffii. Int. J. Syst. Bacteriol. 36:228-240.
16. Buxton, A. E., R. L. Anderson, D. Werdegar, and E. Atlas. 1978. Nosocomial respiratory tract infection and colonization with Acinetobacter calcoaceticus. Am. J. Med. 65:507-513.
17. Cariquist, J. F., M. Conti, and J. P. Burke. 1982. Progressive resistance in a single strain of Acinetobacter calcoaceticus recovered during a nosocomial outbreak. Am. J. Infect. Control 10:43-48.
18. Castle, M., J. H. Tenney, M. P. Weinstein, and T. C. Eickhoff. 1978. Outbreak of a multiple resistant Acinetobacter in a surgi- cal intensive care unit: epidemiology and control. Heart Lung 7:641 644.
19. Cefai, C., J. Richards, F. KL Gould, and P. McPeake. 1990. An outbreak of Acinetobacter respiratory tract infection resulting from incomplete disinfection of ventilatory equipment. J. Hosp. Infect. 15:177-182.
20. Coderre, P. E., and C. F. Earhart. 1984. Characterization of a plasmid carrying the Escherichia coli K12 entD, fepA, fes, and entF genes. FEMS Microbiol. Lett. 25:111-116.
21. Cox, C. D., K. L. Rinehart, Jr., L. Moore, and J. C. Cook, Jr. 1981. Pyochelin: novel structure of an iron-chelating growth promoter from Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 78:4256-4260.
22. Crosa, J. H. 1980. A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an iron- sequestering system. Nature (London) 28:566-568.
23. Crosa, J. H. 1989. Genetics and molecular biology of sidero- phore-mediated iron transport in bacteria. Microbiol. Rev. 53:517-530.
24. Crosa, J. H., and L. L. Hodges. 1981. Outer membrane proteins induced under conditions of iron limitation in the marine fish pathogen Vibrio anguillarum 775. Infect. Immun. 31:223-227.
25. Crosa, J. H., L. K. Luttropp, and S. Falkow. 1978. Molecular cloning of replication and incompatibility regions from the R plasmid R6K. J. Mol. Biol. 124:443-468.
26. Crosa, L. M., M. K. Wolf, L. A. Actis, J. Sanders-Loehr, and J. H. Crosa. 1988. New aerobactin-mediated iron uptake system
in a septicemia-causing strain of Enterobacter cloacae. J. Bac- teriol. 170:5539-5544.
27. Csaky, T. 1948. On the estimation of bound hydroxylamine. Acta Chem. Scand. 2:450-454.
28. Demange, P., A. Bateman, A. Dell, and M. Abdaliah. 1988. Structure of azotobactin D, a siderophore of Azotobacter vine- landii strain D (CCM289). Biochemistry 27:2745-2752.
29. Demange, P., A. Bateman, C. Mertz, A. Dell, Y. Piemont, and M. Abdaliah. 1990. Bacterial siderophores: structures of py- overdins Pt, siderophores of Pseudomonas tolaasii NCPPB2192, and pyoverdins Pf, siderophores of Pseudomonas fluorescens CCM2789. Identification of an unusual natural amino acid. Biochemistry 29:11041-11051.
30. Devaud, M., F. H. Kayser, and B. Bachi. 1982. Transposon- mediated multiple antibiotic resistance inAcinetobacter strains. Antimicrob. Agents Chemother. 22:323-329.
31. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13.
32. French, G. L., M. W. Casewell, A. J. Roncoroni, S. Knight, and I. Phillips. 1980. A hospital outbreak of antibiotic-resistant Acinetobacter anitratus: epidemiology and control. J. Hosp. Infect. 1:125-131.
33. Goldstein, F. W., A. Labigne-Roussel, G. Gerbaud, C. Carlier, E. Collatz, and P. Courvalin. 1983. Transferable plasmid-medi- ated antibiotic resistance inAcinetobacter. Plasmid 10:138-147.
34. Griffiths, E. 1987. The iron-uptake systems of pathogenic bac- teria, p. 69-137. In J. J. Bullen and E. Griffith (ed.), Iron and infection. John Wiley Ltd., New York.
35. Griffiths, G. L., S. P. Sigel, S. M. Payne, and J. B. Neilands. 1984. Vibriobactin, a siderophore from Vibrio cholerae. J. Biol. Chem. 259:383-385.
36. Guenther, S. H., J. 0. Handley, and R. P. Wenzel. 1987. Gram-negative bacilli as nontransient flora on the hands of hospital personnel. J. Clin. Microbiol. 25:488-490.
37. Hancock, R. E. W., K. Hantke, and V. Braun. 1977. Iron transport in Escherichia coli K-12: 2,3-dihydroxybenzoate-pro- moted iron uptake. Arch. Microbiol. 114:231-239.
38. Hantke, K. 1990. Dihydroxybenzolyserine-a siderophore of E. coli. FEMS Microbiol. Lett. 67:5-8.
39. Hartstein, A. I., L. Rashad, J. M. Liebler, L. A. Actis, J. Freeman, J. W. Rourke, T. S. Stibolt, M. E. Tolmasky, G. E. Ellis, and J. H. Crosa. 1988. Multiple intensive care unit outbreak of Acinetobacter calcoaceticus subspecies anitratus respiratory infection and colonization associated with contam- inated, reusable ventilator circuits and resuscitation bags. Am. J. Med. 85:624-631.
40. Joly-Guillou, M. L., Bergogne-Berezin, E., and J. F. Vieu. 1990. Epidemiology of Acinetobacter and resistance to antibiotics at hospitals. A 5-year evaluation. Presse Med. 19:357-361.
41. Lambert, T., G. Gerbaud, P. Bouvet, J. F. Vieu, and P. Courvalin. 1990. Dissemination of amikacin resistance gene aphA6 in Acinetobacter spp. Antimicrob. Agents Chemother. 34:1244-1248.
42. Larson, E. 1984. A decade of nosocomialAcinetobacter. Am. J. Infect. Control 12:14-18.
43. Larson, E. L. 1981. Persistent carriage of gram-negative bacteria on hands. Am. J. Infect. Control 9:112-119.
44. Lemos, M. L., P. Salinas, A. E. Toranzo, J. L. Barja, and J. H. Crosa. 1988. Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum. J. Bacteriol. 170: 1920-1925.
45. Meade, H. M., S. R Long, G. V. Ruvkum, S. E. Brown, and F. M. Ausubel. 1982. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti in- duced by transposon TnS mutagenesis. J. Bacteriol. 149:114- 122.
46. Moffet, H. L., and T. Williams. 1967. Bacteria recovered from distilled water and inhalation therapy equipment. Am. J. Dis. Child. 114:7-12.
47. Morgan, M. E. I., and C. A. Hart. 1982. Acinetobacter menin- gitis: acquired infections in a neonatal intensive care unit. Arch. Dis. Child. 57:557-559.
J. BACTERIOL.
D ow
nl oa
de d
fr om
h ttp
s: //j
ou rn
al s.
as m
.o rg
/jo ur
na l/j
b on
1 6
D ec
em be
r 20
21 b
y 19
1. 24
0. 11
9. 20
IRON ACQUISITION IN A. BAUMANNII 7679
48. Murray, B. E., and R. C. Moellering, Jr. 1980. Evidence of plasmid-mediated production of aminoglycoside-modifying en- zymes not previously described in Acinetobacter. Antimicrob. Agents Chemother. 17:30-36.
49. Obana, Y. 1986. Pathogenic significance of Acinetobacter cal- coaceticus: analysis of experimental infection in mice. Micro- biol. Immunol. 30:645-657.
50. Ong, S. A., T. Peterson, and J. B. Neilands. 1979. Agrobactin, a siderophore from Agrobacterium tumefaciens. J. Biol. Chem. 254:1860-1865.
51. Page, W. J., and M. Huyer. 1984. Derepression of the Azoto- bacter vinelandii siderophore systems using iron-containing minerals to limit iron repletion. J. Bacteriol. 158:496-502.
52. Persmark, M., D. Expert, and J. B. Neilands. 1989. Isolation, characterization, and synthesis of chrysobactin, a compound with siderophore activity from Erwinia chrysanthemi. J. Biol. Chem. 264:3187-3189.
53. Persmark, M., T. Frejd, and B. Mattiasson. 1990. Purification, characterization, and structure of pseudobactin 589A, a sidero- phore from plant growth promoting Pseudomonas. Biochemis- try 29:7348-7356.
54. Pickett, C. L., L. Hayes, and C. F. Earhart. 1984. Molecular cloning of the Escherichia coli K12 ent ACGBE genes. FEMS Microbiol. Lett. 24:77-80.
55. Pollack, J. R., B. N. Ames, and J. B. Neilands. 1970. Iron transport in Salmonella typhimurium: mutants blocked in the biosynthesis of enterobactin. J. Bacteriol. 104:635-639.
56. Ramphal, R., and R M. Kluge. 1979. Acinetobacter calcoace- ticus variety anitratus: an increasing nosocomial problem. Am. J. Med. Sci. 277:57-66.
57. Retailliau, H. F., A. W. Hightower, R. E. Dixon, and J. R. Allen. 1979. Acinetobacter calcoaceticus: a nosocomial pathogen with unusual seasonal pattern. J. Infect. Dis. 139:371-375.
58. Rogers, H. J. 1973. Iron-binding catechols and virulence in Escherichia coli. Infect. Immun. 7:445-456.
59. Rosenthal, S., and I. B. Tager. 1975. Prevalence of gram- negative rods in the normal pharyngeal flora. Ann. Intern. Med. 83:355-357.
60. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
61. Schryvers, A. B., and L. J. Morris. 1988. Identification and characterization of the transferrin receptor of Neisseria menin- gitidis. Mol. Microbiol. 2:281-288.
62. Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160:47-56.
63. Simoni, R. D., and P. W. Postma. 1975. The energetics of bacterial active transport. Annu. Rev. Biochem. 44:523-554.
64. Simpson, L. M., and J. D. Oliver. 1983. Siderophore production by Vibno vulnificus. Infect. Immun. 41:644-649.
65. Smith, A. W., and K. E. Alpar. 1991. Immune response to Acinetobacter calcoaceticus infection in man. J. Med. Micro- biol. 34:83-88.
66. Smith, A. W., S. Freeman, W. G. Minett, and P. A. Lambert. 1990. Characterization of a siderophore from Acinetobacter calcoaceticus. FEMS Microbiol. Lett. 70:29-32.
67. Smith, P. W., and R. M. Massanari. 1979. Room humidifiers as the source ofAcinetobacter infections. JAMA 237:795-797.
68. Southern, E. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.
69. Tolmasky, M. E., P. A. Salinas, L. A. Actis, and J. H. Crosa. 1988. Increased production of the siderophore anguibactin me- diated by pJM1-like plasmids in Vibrio anguillarum. Infect. Immun. 56:1608-1614.
70. Valdez, J. M., M. 0. Asperilla, and R. A. Smego, Jr. 1991. Acinetobacter peritonitis in patients receiving continuous am- bulatory peritoneal dialysis. South. Med. J. 84:607-610.
71. van Tiel-Menkveld, G. J., A Rezee, and F. K. DeGraaf. 1979. Production and excretion of cloacin DF13 by Escherichia coli harboring plasmid CloDF13. J. Bacteriol. 140:415-423.
72. Williams, P. H. 1979. Novel iron uptake system specified by ColV plasmids, an important component in the virulence of invasive strains of Escherichia coli. Infect. Immun. 29:411-416.
73. Wise, KL A., and F. A. Tosolini. 1990. Epidemiological surveil- lance ofAcinetobacter species. J. Hosp. Infect. 16:319-329.
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