Resistance to bismuth among Gram-negative bacteria is dependent upon iron and its uptake

Journal of Antimicrobial Chemotherapy (1996) 38, 1031-1040

Resistance to bismuth among Gram-negative bacteria is dependentupon iron and its uptake

Philip Domenico, Jay Reich, William Madonia and Burke A. Cunha

Infectious Disease Division, Winthrop-University Hospital, Mineola, Long Island,New York 11501 and SUNY School of Medicine, Stony Brook, NY, USA

Bismuth antimicrobial action is poorly understood. Many tnvalent metals possessantibacterial activity, especially under low iron conditions. Protection of bacteriafrom the deleterious effects of bismuth and other trivalent metals was demonstratedin iron-fortified media. Near-equimolar quantities of Fe3"1" neutralized thegrowth-inhibitory effects of 250 /IM Bi3+. Resistance to bismuth action also dependedon the production of virulence-related siderophores. Escherichia coli, Aeromonashydrophila or Pseudomonas aeruginosa producing aerobactin, amonabactin orpyoverdin respectively, were most resistant to Bi3+. Enterochelin or pyochelinproducers were less resistant to Bi3+, but more resistant than strains lackingsiderophores. Purified pyoverdin restored Bi3+ resistance in a mutant lacking thissiderophore, but not in one lacking the pyoverdin receptor. Bismuth-treated bacteriaexhibited unique outer membrane proteins, similar in size to iron-repressible proteins.Thus, resistance to the inhibitory action of Bi3+ among Gram-negative bacteria isinversely related to iron concentration and strongly dependent on iron transportmechanisms. The data suggest that bismuth action is largely a nonspecific,competitive interference with iron transport, related primarily to atomic valence.Furthermore, resistance to Bi3+ among bacteria is predictive of virulence.

Introduction

Iron is necessary for proliferation of bacteria in host tissues (Weinberg, 1978). Bacteriahave developed elaborate means to wrest iron from the environment. Pathogenicbacteria often possess high-affinity iron-transport systems, which rely on siderophoresto scavenge iron, and transport systems that actively sequester iron intracellularly(Neilands, 1981a). Most members of the family Enterobacteriaceae produce acatechol-type siderophore enterobactin, which has been implicated in virulence (Yancey,Breeding & Lankford, 1979). A number of strains also produce aerobactin, ahydroxamate-type siderophore (Neilands, 1981a), which is the siderophore most oftenassociated with virulence (Williams & Warner, 1980; Konopka, Bindereif & Nielands,1982; de Lorenzo & Martinez, 1986; Nassif & Sansonetti, 1986; Nassif et al., 1987).

The two best-studied siderophores among the pseudomonads are pyochelin andpyoverdin, both of which impart pigmentation to pseudomonas colonies. Likeaerobactin among enteric bacteria, pyoverdin is the 'virulence' siderophore among

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pseudomonads. Pyochelin mutants grow in human sera, whereas pyoverdin mutants donot (Ankenbauer, Sriyosachati & Cox, 1985). Among Aeromonas species, amonabactinis the virulence-associated siderophore, while most species excrete enterobactin for ironscavenging (Massad, Arceneaux & Byers, 1991).

Though bismuth-containing remedies have been in regular use for centuries, onlyrecently has Bi3+ been taken seriously as an antimicrobial agent (Sox & Olson, 1989;Manhart, 1990). The antimicrobial effects of Bi3+ span a wide range of bacteria,affecting inhibition of bacterial growth at 2-10 mM, levels deemed therapeuticallyachievable in the digestive tract (Cornick, Silva & Gorbach, 1990; Manhart, 1990).However, little is known about its mechanism of action. Antibacterial effects of othertrivalent metals have been linked to iron. Inhibition of bacterial growth by indium(In3+), gallium (Ga3+), and aluminum (Al3+) occurred especially during iron-limitedgrowth (Fekete & Barton, 1991). The antimicrobial action of Ga3+ was abolished byexcess Fe3+ in culture medium (al Aoukaty, Appanna & Falter, 1992). Alumimum,chromium (Cr3+), or Ga3+ ions do not displace Fe3+ from siderophores except underreducing conditions (Emery, 1986). Pseudomonads overproduce pseudobactin underiron-limitation in the presence of 1 mM of one of several trivalent metals, including Cr3+,scandium (Sc3+), and yttrium (Y3+).

The level of Fe3+ in culture media also affects the inhibitory action of Bi3+. The aimof this report is to characterize the influence of Fe3+ and its transport mechanisms onBi3+ action among Gram-negative bacteria.

Methods

Bacterial strains and cultivation

A quantitatively-defined salts broth medium (M9) was used to culture Aeromonashydrophila and Escherichia coli. Pseudomonas aeruginosa was cultured in succinateminimal medium (Cox & Adams, 1985). The bacteria are listed in the Table. Brothmedium was made iron-limiting by using acid-washed glassware, and by treatingphosphate buffer stock solutions with Chelex 100. Media and other solutions wereprepared in distilled, deionized water. Starter cultures, grown to mid-log phase at 35°Cfor 4 h on an orbital shaker at 200 rpm, were used to prepare 0.5 MacFarland standardsuspensions, which were further diluted 1:100 (5 x 103cfu/mL) into test medium.Cultures were incubated and monitored for turbidity in an A vantage MicrobiologySystem (Abbott Laboratories, Irving, TX, USA). Viable bacterial counts (cfu/mL) andsubculturing were performed by standard plating on nutrient agar (BBL, Cockeysville,MD, USA).

Biochemicals

Stock solutions of 100 mM Bi(NO3)3 (Sigma Chemical Co., St. Louis, MO, USA) wereprepared fresh daily in propylene glycol containing 400 mM NaOH, pH 12.0. Ferric andferrous chloride (Sigma) were prepared weekly in distilled, deionized water. The ironchelator 2,2'-dipyridyl was obtained from Sigma. All solutions were kept at 4°C.Iron-free pyoverdin was recovered by ethyl acetate extraction from culture supernatantsof iron-limited P. aeruginosa PAO1 (Poole, Neshat & Heinriks, 1991).


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Bismuth-iron antagonism among bacteria 1033

Table. Bacterial strains

Strain

E. coliD551LG1695H1443HI 594C10O1C1072C1087C1115

A. hvdrophila495A2SB22279279.3K

P. aeruginosaK.199 (PAO1)K.201 (PAO6609)

K394

Description

O78:H-, plasmid-encoded aerobactinplasmid-free derivative of D551araD 139 A/acl69 aroB rspl thiAs H1443 butfiu::Mu d l XJiu::Mu dl(ap lac) cirAs HI443 but tonBcirfepA cir fiu

amonabactin producingamonabactin deficient (495A2 derivative)enterobactin producingenterobactin deficient (279 derivative)

wild typemet -9011 amiE200 rspl pyd-9As K.199, but pyoverdin deficientAs K201, but pyochelin deficient; noferripyochelin or ferripyoverdin receptor

Source or reference

Roberts el al. (1989)Roberts et al. (1989)Hantke (1983)Hankte (1983)Curtis et al. (1988)Curtis et al. (1988)Curtis et al. (1988)Curtis et al. (1988)

Massad et al. (1991)Massad et al. (1991)Massad el al. (1991)Massad et al. (1991)

Poole et al. (1991)Hohnadel,

Hasse & Mayer (1986)Poole et al. (1991)

Assay conditions

Varying concentrations of Bi(NO3)3 and FeCl3 were added to broth medium. Bacterialcultures were monitored for growth in the Avantage Microbiology System (Abbott,Chicago, IL, USA), which gently agitated cultures at 35°C and recorded turbiditymeasurements every 5 min. Growth inhibition was determined by monitoring lag timesfrom inoculation to initiation of culture growth (Domenico, Landolphi & Cunha, 1991),and by standard plating in triplicate at 18 h growth. Culture lag times were measuredfrom computer-generated growth curves.

Characterization of iron-repressible outer membrane proteins {IROMPs)

Broth cultures were grown to exponential-phase in the presence of 100 (MM 2,2'-dipyridylor Bi(NO3)3 at 250 /iM in iron-poor medium. Bacteria were harvested by centrifugationat 4°C, resuspended in 0.1 M LiCl and shaken vigorously with glass beads for one hourat room temperature to obtain outer membrane fragments, which were isolated bydifferential centrifugation. Sarkosyl-insoluble outer membrane fractions were analysedby SDS-PAGE (Laemmli, 1970) with silver staining (BioRad, Richmond, CA, USA)to detect Bi3+-inducible, iron-repressible proteins.

Results

Iron was shown to protect a number of Gram-negative bacteria from Bi3+ action.Among A. hydrophila, resistance to Bi3+ depended on the siderophore produced. Underiron limitation, growth in the absence of bismuth produced only minor differences inculture lag times for an amonabactin-producing strain, an amonabactin-deficientmutant, a strain producing only enterobactin, or an enterobactin-deficient mutant (data


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1034 P. Domenieo et al.

1500

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0Added ferric chloride (log (|!M))

Figure 1. Siderophore-related bismuth resistance among A. hydrophila. Four Aeromonas strains werecultured in iron-limited defined medium containing 250 im (Bi(NO))j. Strains included an amonabactinproducer ( • ) and its isogenic, amonabactin-deficient mutant (#), an enterobactin producer (A), and itsisogenic, enterobactin-deficient mutant ( • ) . Added to the culture medium were varying amounts of FeJ+ tostimulate bacterial growth Cultures were monitored for turbidity. Culture lag times were determined fromgrowth curves (error bars represent one standard deviation).

1x10

1x10'0 20 40 60 80 100 120 140 160 180 200 220 240 260

Added ferric iron (|iM)

Figure 2. Siderophore-related bismuth resistance among E. colt. Two E colt strains were cultured iniron-limited defined medium with or without 250 IXM BI(NOJ)J. Cultures included an aerobactin producer with(D) or without ( • ) bismuth added, and its isogenic, aerobactin-deficient mutant with (A) or without (A)bismuth added. Ferric iron was added to culture medium in varying amounts to stimulate bacterial growth.Viable cells were quantified at 18 h growth by standard plating in triplicate (error bars represent one standarddeviation).


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not shown). However, in the presence of 250/iM Bi(NO3)3 (Figure 1), the amonabactinstrain exhibited a marked growth advantage, particularly in iron-limited medium. Thestrain producing enterobactin had only a slight growth advantage over its mutant.Addition of equimolar Fe3+ decreased the lag time for all Bi3+-treated strains to nearnormal.

Siderophore-based resistance to bismuth was also seen in E. coli. Strain D551produced both aerobactin and enterobactin, while its derivative, strain LG1695produced enterobactin only. In iron-poor medium, strain D551 showed a significantgrowth advantage over strain LG1695 at 18 h of incubation (Figure 2). At 1.5 //M iron,growth of strain D551 was near optimal, with respect to bacterial count, while nearly100/iM Fe3+ was required for LG1695 optimum growth. These bacteria differedmarkedly in their susceptibility to Bi3+. Growth of D551 was not significantly affectedby 250 (iM (Bi(NO3)3, even in limiting iron. In contrast, LG1695 was strongly inhibitedby the same Bi3+ concentration, and required equimolar Fe3+ to reverse the inhibition.

E. coli iron receptor mutants were also examined for susceptibility to Bi3+. Inhibitoryconcentrations of Bi3+ ranged from 1.5 to 6 mM. No particular strain showed notablygreater or lesser sensitivity to Bi3+ than the parent strain, although a tonB mutant wasslightly more sensitive to Bi3+ than were fepA, cir,fiu, or cir fiu mutants (data notshown).

Resistance to Bi3+ was also assessed in P. aeruginosa pyoverdin mutants. The quantityof Fe3+ needed to stimulate growth of bacteria treated with 250 HM (Bi(NO3)3 differedamong strains depending on their iron uptake mechanisms (Figure 3). Purifiedpyoverdin was added to growth medium to produce a fluorescence similar in pigmentintensity to that of overnight K199 cultures (A^o™ = 3.7). Under limiting iron, the

lxio

1x10'-3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5

Added ferric iron (log(|iM))2 2.5 3

Figure 3. Effect of purified pyoverdin on resistance of P. aeruginosa to Bi3 + . Three related strains of P.aeruginosa were grown in culture medium containing 250 /iM bismuth. Purified pyoverdin was added to themedium in some trials. Ferric iron was added to culture medium in varying amounts to stimulate bacterialgrowth. Viable cells were quantified at 18 h growth by standard plating in triplicate (error bars represent onestandard deviation). Bacterial strains: KI99 (D); K199 + pyoverdin ( • ) ; K20I (O); K20I + pyoverdin ( • ) ;K394 (A); K394 + pyoverdin (A)


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A B C D

kDal

116

97

66

48.5

F G H I J K L M

Figure 4. SDS-PAGE of E coli IROMPs. Outer membrane protein samples from broth cultures of E. coliiron uptake mutants were electrophoresed on a 12% separating gel and silver-stained. Molecular weightmarkers are shown in lane A. Proteins in lanes B (control) and C (100/(M Bi' + ) were obtained from strainH1443. Proteins in lanes D (control) and E (100 /iM Bi3 + ) were obtained from strain H1594 Proteins in lanesF (control) and G (100/JM Bi'*) were obtained from strain C100I. Proteins in lanes H (control) and I (100/JMBi1* ) were obtained from strain C1072 Proteins in lanes J (control) and K (100 /JM BI3 + ) were obtained fromstrain C1087 Proteins in lanes L (control) and M (100/JM BI3 + ) were obtained from strain CI115. Arrowsindicate novel proteins in bismuth-treated samples.

bacterial count was two logs higher in K.199 than in pyoverdin-deficient K201 cultures.Added pyoverdin produced a 0.5 and 1.5 logm increase in bacterial count for K199 andK.201, respectively. Equimolar Fe3 + restored growth to optimal levels. Iron limitationproduced a more profound impact on strain K394, a K201-derived strain lacking thepyoverdin receptor. Pyoverdin did not enhance K394 growth regardless of Fe3 +

concentration.Outer membrane proteins were also altered by treatment with Bi3 + . SDS-PAGE

protein profiles from Bi3 +-treated bacteria are presented for E. coli iron transportmutants (Figure 4) and for A. hydrophila (Figure 5). Protein profiles from E. coli strainsrevealed two new proteins with apparent molecular weights of 66 and 77 kDa. Theirelectrophoretic mobilities are consistent with other IROMPs, but they appear unrelatedto cir,Jiu, or fepA. A unique 71 kDa protein band was seen in profiles of A. hydrophila.


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kDal

116

B C

1 I[f]

66

48.5 mm

29

Figure 5. SDS-PAGE of A. hydrophila IROMPs. Outer membrane protein samples from broth cultures wereelectrophoresed on a 12% separating gel and silver stained. Molecular weight markers are shown in LaneA. Proteins in Lane B were from iron-replete (50 nu Fe! + ) cultures. Proteins in Lane C were fromiron-depleted (100 //M 2, 2'-dipyndyl) cultures. The I ROMP region is marked by brackets. Lanes D containsproteins from iron-replete cultures treated with 250//M bismuth nitrate. Arrow indicates novel protein inbismuth-treated samples.

again within the IROMP region. The novel proteins were not identified with knownIROMPs, and were not characterized further.

Discussion

This report documents the competitive relationship between iron and bismuth amongGram-negative bacteria. Both iron concentration and uptake mechanisms play a role


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in resistance to bismuth. Thus, in iron-depleted media, low Bi3+ concentrations inhibitedbacterial growth. Bismuth is similar in this regard to other trivalent metals (Emery,1986; Fekete & Barton, 1991; al Aoukaty et al., 1992), suggesting that the effect onbacteria is nonspecific, and related primarily to atomic valence.

It is considered unlikely that trivalent metals compete well with Fe3+ (Neilands,19816). However, iron transport mechanisms were shown here to influence strongly thedegree of competition. Certain siderophores, especially those associated with virulence(i.e., aerobactin, pyoverdin, amonabactin), impart resistance to Bi3+, presumably dueto their high selectivity for Fe3+. Strains producing enterobactin were not as resistantto Bi3+ as strains producing aerobactin or amonabactin, but were generally moreresistant than siderophore-deficient strains. Deficiencies in siderophore synthesis lead toBi3+-hypersensitivity.

The inhibitory effect of Bi3+ appears to be due largely to interference with irontransport. In effect, Bi3+ toxicity may simply be iron starvation. Extracellularcompetition with Fe3+ for siderophores or ferric iron receptors is likely to be onemechanism of Bi3+ and other trivalent metal action. Bismuth may simply coat thebacterial surface, thereby blocking Fe3+ receptors. Alternatively, the Fe3+/Bi3+

competition could occur in the cytoplasm. The high affinity of Bi3+ for thiol groups mayinterfere with iron-sulphur centres of enzymes involved in ATP synthesis (Beil et al.,1995). Accumulation of Bi3+ by bacteria could create a functional iron limitation in thecytoplasm, which in turn could signal IROMP induction.

Several unique proteins were noted in SDS-PAGE of outer membrane preparations.The proteins (66 and 77 kDa in E. coli and 71 kDa in A. hydrophila) are in the IROMPregion, which supports their identity as receptors for iron transport. By definition theyare Bi3+-inducible, iron-repressible outer membrane proteins, and may be unrelated toother IROMPs. Some doubt remains as to whether these are true outer membraneproteins, or whether their gel mobility is related to their molecular size. SDS-PAGE ofsamples from Bi3+-treated cultures proved difficult, often resulting in smeared profiles,possibly due to cell damage. Bismuth also binds to proteins, and this may alter theirelectrophoretic mobility (Slikkerveer & DeWolff, 1989), though no mobility effects wereobserved with known molecular markers.

Iron-mediated resistance to trivalent metals sheds light on their mechanism of action.Iron is a cofactor in numerous enzymatic processes involving oxidation-reductionreactions that produce energy for the cell. The importance of cytochromes andnon-heme iron in the respiratory chains of aerobic and facultative bacteria underscoresthe role of iron in energy metabolism (Neilands, 1981a). By blocking transport of ferriciron, or by binding to enzyme thiol groups, Bi3+ could inhibit enzyme function indirectlyor directly. Inhibition of oxidative processes serves to deplete intracellular energy levels.Bacteria react to lowered energy reserves by shutting down nonessential,energy-intensive processes, like capsular polysaccharide biosynthesis (Domenico et al.,1991) and production of fimbriae (Kunin et al., 1994). Higher bismuth levels producesevere depletion of energy reserves and interfere with cell growth (Domenico et al.,1991). Indeed, levels of ATP in bacteria are decreased by 90% in the presence of 10 mMbismuth subsalicylate (Sox & Olson, 1989).

As with other antimicrobial agents, continued use of Bi3+ in the gastrointestinal tractmay select for resistant bacteria. Bacteria with superior means of acquiring iron in vivomay be selected during Bi3+ therapy. Colonization by E. coli of the mouse intestineis enhanced by an aerobactin-iron uptake system in the bacteria (Stojiljkovic, Cobeljic


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& Hantke, 1993). Such bacteria have higher invasive potential, and are associated withsystemic infection. Long-term bismuth use could promote colonization by invasivebacteria. Though not substantiated by clinical studies, this possibility should becarefully evaluated, given the popularity of oral Bi3+ therapy. In the laboratory, Bi3+

could be used to select for virulent bacteria in iron-poor media.The conversion to ferrous iron (Fe2 + ) in the anaerobic gut may further limit Bi3+

therapy. While Fe3+ is insoluble, Fe2+ is very soluble and is favoured by gut bacteria(Stojiljkovic et al., 1993). In contrast, Bi3+ is largely insoluble in the anaerobic gut(Bierer, 1990). Thus, Bi3+ potency is diminished in the gut, given the abundance andavailability of Fe2 + . This explains why Bi3+ activity in vitro does not correlate with thatin vivo. Indeed, maximum dosing of Bi3+ did not affect the quantity of bacteria foundin faecal samples (Gorbach, Cornick & Silva, 1990).

With regard to Bi3+ therapy, there are several evolving considerations. Ironacquisition is essential for growth of bacteria in serum and for their virulence in mice(Yancey et al., 1979; Nassif et al., 1987). Competition with Fe3+ may be the primarymeans by which bismuth compounds inhibit bacteria, and awareness of Bi3+/Fe3+

antagonism may foster more effective pharmaceutical preparations. Studies are neededto determine whether dietary iron should be avoided during therapy with Bi3+.Combinations with metal chelators may also influence therapy, and several possibilitiesare currently under investigation.

AcknowledgementsThese studies were sponsored in part by a grant from Procter & Gamble. Special thanksto Peter Wu for his technical assistance.

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(Received 15 December 1995; returned 19 February 1996: revised 27 March 1996: accepted 22 July 1996)


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Resistance to bismuth among Gram-negative bacteria is dependent upon iron and its uptake

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