dactinomycin, sodium dodecyl sulfate, and Triton X-100). A concentration of 0.3% HMP decreased the MICs of the probes by a factor of approximately 10, and ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1989, p. 1741-1747 0066-4804/89/101741-07$02.00/0
Vol. 33, No. 10
Sodium Hexametaphosphate Sensitizes Pseudomonas aeruginosa, Several Other Species of Pseudomonas, and Escherichia coli to Hydrophobic Drugs MARTTI VAARA* AND JOUNI JAAKKOLA Department of Bacteriology and Immunology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki, Finland Received 24 April 1989/Accepted 25 July 1989
Many gram-negative bacteria are known to be remarkably resistant to hydrophobic noxious agents by virtue of their outer membranes (OM). We investigated, by using four different assay methods, the ability of sodium hexametaphosphate (HMP) to disrupt this OM barrier. (i) In the growth inhibition assay, HMP was found to sensitize strains of Pseudomonas aeruginosa to all the hydrophobic probes tested (rifampin, fusidic acid, dactinomycin, sodium dodecyl sulfate, and Triton X-100). A concentration of 0.3% HMP decreased the MICs of the probes by a factor of approximately 10, and maximally even a 30-fold sensitization was found with 1% HMP. (ii) In the bactericidal assay, 0.3% HMP decreased the MBC of the hydrophobic probe rifampin by a factor of approximately 30. (iii) In the bacteriolytic assay, 0.1% HMP sensitized the target bacteria to lysis by sodium dodecyl sulfate and Triton X-100. (iv) In the fluorescent-probe binding assay, HMP drastically enhanced the binding of fluorescent N-phenyl naphthylamine to the membranes of the target cells. In addition to P. aeruginosa, P. fluorescens, P. putida, P. fragi, and Escherichia coli were susceptible to the OM permeability-increasing action of HMP, while P. cepacia was resistant.
Growth temperatures. In all cultivations, P. aeruginosa, P. cepacia, and E. coli were incubated at 37°C, and the other strains were incubated at 28°C. Chemicals. HMP (pure technical grade) was from Allbright & Wilson, London, England). A 10% stock solution (wt/wt) was prepared in deionized water and neutralized (pH 7.0) with NaOH. Triton X-100 and sodium dodecyl sulfate (SDS) were from BDH Ltd., Poole, England. Fusidic acid (sodium
Polyphosphates are highly anionic condensation products of phosphoric acids and form chelate complexes with multivalent cations such as magnesium, calcium, and manganese ions (24, 25). As antimicrobial agents, they are weak or very weak and mainly active against gram-positive bacteria (11). Therefore, they are not generally regarded as antimicrobial agents. On the other hand, it has long been known that EDTA, another metal chelator, has an important antibacterial action: it damages the outer membrane (OM) of gram-negative bacteria and increases its permeability to other antimicrobial agents, such as lysozyme, hydrophobic antibiotics and dyes, and detergents (8, 10). This is a very remarkable property because, in the absence of EDTA, those bacteria are very resistant to the drugs mentioned above (10). Besides EDTA, probably also many other chelators have a similar effect, although much less is known thus far about their action. In this report, we show that sodium hexametaphosphate (HMP), a phosphate polymer, is a potent agent for sensitizing Pseudomonas strains as well as Escherichia coli to hydrophobic drugs and detergents.
salt), rifampin, dactinomycin, and N-phenyl naphthylamine (NPN) were from Sigma Chemical Co., St. Louis, Mo. The stock solution of rifampin was prepared by dissolving 10 mg of rifampin with 1 ml of methanol, after which deionized water was added to a final volume of 10 ml. The following polyanions were from Sigma: phytic acid (dodecasodium salt, P-3168), sodium polyaspartate (P-5387; degree of polymerization, approximately 84), sodium polyglutamate (P-4636; degree of polymerization, approximately 88), and sodium polygalacturonate (P-1879). Polymyxin B nonapeptide (PMBN) was prepared essentially as described by Viljanen and Vaara (27) and was a kind gift from Farmos Group Ltd., Turku, Finland. Its residual content of polymyxin B was approximately 0.1%, as determined by reversed-phase high-pressure liquid chromatography (19). EDTA (disodium salt) was from E. Merck AG, Darmstadt, Federal Republic of Germany. The stock solution (100 mM) was neutralized (pH 7.0) with NaOH. Growth inhibition assay. Bacterial colonies grown overnight on L agar were suspended in medium B to a final absorbance of 60 Klett units (Klett-Summerson colorimeter, red filter; corresponding to ca. 0.5 x 109 bacteria per ml). Further dilutions of this suspension (in medium B) were used to inoculate (with ca. 104 cells per ml) the assay media containing increasing concentrations of the hydrophobic probe compound (e.g., rifampin). Samples (100 1.l) of these inoculated media were pipetted into wells of a microdilution plate (Nunclon Delta, catalog no. 167008; Nunc, Roskilde, Denmark). The wells already contained increasing amounts of HMP (or PMBN or EDTA) in 10 ,ul of water. The plates
MATERIALS AND METHODS Bacterial strains. Pseudomonas aeruginosa strains included PAO1 (4) as well as ATCC 27577 (international serotype 3) and ATCC 27584 (international serotype 6). Other Pseudomonas strains were P. cepacia ATCC 25609, P. fragi ATCC 4973, P. putida ATCC 12633, and P. fluorescens ATCC 17634. E. coli IH3080 (20) is a smooth, encapsulated 018:K1 strain. Growth media. L broth contained 10 g of tryptone (Difco Laboratories, Detroit, Mich.), 5 g of yeast extract (Oxoid Ltd., Hampshire, United Kingdom), and 5 g of NaCl per liter and was adjusted to pH 7.2. L agar also contained 1.5% agar. Media A, B, and C were 1:1, 1:2, and 1:4 mixtures (vol/vol), respectively, of L broth and deionized water. *
Corresponding author. 1741
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VAARA AND JAAKKOLA
were incubated at the growth temperature (see above) for 18 h, after which the A405 of each well was measured with a Titertek Multiscan spectrophotometer (Labsystems, Helsinki, Finland). Before the reading, the spectrophotometer was blanked with corresponding uninoculated assay media. Measuring the bactericidal effect. The bactericidal effect was measured essentially by the method reported previously by our laboratory (26). Bacteria were grown in L broth on a
rotary shaker at 220 rpm and 37°C to the early logarithmic growth phase (60 Klett units). These bacteria were used to inoculate (with 5 x 104 cells per ml) the assay media (medium B containing increasing concentrations of rifampin). Samples (100 ,u) of these inoculated media were pipetted into microdilution wells (see above) which already contained increasing amounts of HMP. The plates were incubated at 37°C for 2 h, and the viable counts in each well were determined by transferring the entire contents of each well to separate vials, followed by serial dilution of the contents (in 150 mM saline) and plating onto L agar. The colonies were allowed to grow for 24 h at 37°C, and their number was compared with that in the inoculum. Bacteriolysis assays. Bacteria were grown in L broth as described above to the early logarithmic phase of growth (40 Klett units), washed in 0.01 M HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4) containing 50 mM NaCl, and resuspended in the same buffer to a final absorbance of approximately 120 Klett units. Samples (100 RI) of this suspension were then rapidly (in 15 s) pipetted into a total of 24 wells of a microdilution plate (Nunclon Delta). Each well already contained a known amount of HMP as well as SDS or Triton X-100 (in 20 ,l of water). The A405 in each well was then monitored at 1-min intervals with a Titertek Multiscan spectrophotometer. NPN fluorescence experiments. The fluorescence experiments were done essentially as described elsewhere by Uratani (17). A bacterial suspension in 0.01 M HEPES (pH 7.4) with 50 mM NaCl was prepared as in the bacteriolysis assays (see above). To 3-ml samples of such a suspension, increasing concentrations of HMP and a constant amount of NPN (30 ,ul of a 1i-' M solution in methanol) were added, in that order. The fluorescence was then immediately measured with a spectrofluorometer (LS-3; excitation wavelength, 360 nm; emission wavelength, 420 nm; sensitivity setting, 0.1; The Perkin-Elmer Corp., Norwalk, Conn.) equipped with a chart recorder. Background fluorescence in the absence of HMP was subtracted. Separate controls indicated that HMP alone (without bacteria) did not increase the fluorescence of NPN. RESULTS Synergistic growth inhibition. (i) HMP and rifampin against P. aeruginosa PAO1. Rifampin is a typical representative of the large group of hydrophobic antibacterial agents against which the OM is known to be an effective permeability barrier. It does not effectively permeate the intact OM of gram-negative enteric bacteria and P. aeruginosa but traverses through the OM of certain OM-defective mutants as well as through the OM damaged by EDTA (8) or polycations (20, 26, 27). Because these properties of rifampin have been reasonably well characterized, we chose rifampin to be our main probe to detect the HMP-induced damage to the OM. HMP and rifampin proved to act synergistically in inhibiting the growth of P. aeruginosa PAO1 (Fig. 1). Thus, the concentration of rifampin required to reduce the bacterial
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ANTIMICROB. AGENTS CHEMOTHER.
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FIG. 1. Synergistic growth-inhibitory action of HMP and rifampin against P. aeruginosa PAO1. Bacteria were grown for 18 h in the presence of increasing concentrations of HMP and rifampin in medium B (see Materials and Methods) in the wells of a microdilution plate. Then the growth was quantified by measuring the A405 with a multichannel spectrophotometer and compared with the growth of the control (the well which lacked both HMP and rifampin). Each datum point is the mean + standard deviation of six independent determinations.
growth to 1/10 that of the control was approximately 10 ,ug/ml in the absence of HMP but approximately 1 ,ug/ml in the presence of 0.3% HMP. HMP itself (in the absence of rifampin) had a definite, although slight, growth-retarding effect. The findings presented above were obtained by using medium B as the growth medium. Medium B is a 1:2 (vol/vol) mixture of L broth and deionized water (see Materials and Methods). Our preliminary findings (data not shown) indicated that in undiluted L broth the synergistic effect of HMP and rifampin was less pronounced. A clear synergism was seen in medium A (a 1:1 mixture of L broth and water) (Fig. 2A). When L broth was further diluted (medium C, 1:4 mixture), the synergism appeared to be even more marked (Fig. 2C). Thus, in medium C, 1% HMP decreased the MIC of rifampin by a factor of approximately 30. When 5 mM MgCl2 was added into medium A, 0.3% HMP completely lost its ability to sensitize the bacteria to rifampin (data not shown). To assess the significance of the synergism found in this study, we compared the synergistic system of HMP and rifampin with two previously characterized synergistic systems (EDTA and rifampin, and PMBN and rifampin) (Fig. 3). Under the conditions used (medium B), 3 mM EDTA was approximately as effective as 0.3% HMP in sensitizing the target bacteria to rifampin (and 1 mM EDTA was practically ineffective). A concentration of 10 mM EDTA alone (without rifampin) completely inhibited the growth. PMBN, which has a very potent OM permeability-increasing property (10, 21-23, 26, 27), sensitized the bacteria by a factor of approximately 100, even at as low a concentration as 1 jig/ml. In undiluted L broth, the effects of EDTA were much weaker than in medium B, but the effects of PMBN unchanged (data not shown). (ii) HMP and rifampin against other Pseudomonas strains and E. coli. Other P. aeruginosa strains tested (ATCC 27577
HMP-INDUCED SENSITIZATION TO HYDROPHOBIC DRUGS
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(pg/mi)
broth, in which the MIC of rifampin was 10 ,ug/ml in the absence of HMP, 1 ,ug/ml in the presence of 0.3% HMP, and 0.3 ,ug/ml in the presence of 1% HMP (data not shown). (iii) HMP and hydrophobic agents other than rifampin. From the numerous other hydrophobic agents against which
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and 27584) were as susceptible as PAO1 to the synergistic action of HMP and rifampin. Susceptible strains also included P. fragi ATCC 4973 (Fig. 4A), P. fluorescens ATCC 17634 (Fig. 4B), and P. putida ATCC 12633. In contrast, P. cepacia ATCC 25609 was completely resistant to HMP (no growth inhibition at 3 jig of rifampin per ml in the presence of even 1% HMP and full inhibition at 10 ,ug of rifampin per ml both in the absence of HMP and in the presence of 1% HMP). Also E. coli (the smooth, encapsulated 018:K1 strain IH3080) was sensitive to the HMP-rifampin system (Fig. 4C). This sensitivity was also clearly seen in undiluted L
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FIG. 3. OM permeability-increasing action of HMP in comparison with that of EDTA and PMBN. The target organism (P. aeruginosa PA01), the probe compound (rifampin), and the experimental conditions were identical to those used in the experiment illustrated in Fig. 2B.
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VAARA AND JAAKKOLA
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the intact OM acts as a barrier, we included in our study the antibiotics fusidic acid and dactinomycin, as well as the detergents SDS and Triton X-100 (18, 21, 22, 26, 27). HMP acted synergistically with all those agents (Fig. 5; data not shown). (iv) Polyanions other than HMP. Sodium polyglutamate, sodium polyaspartate, sodium galacturonate, and sodium phytate had negligible or very weak synergistic actions with rifampin (tested against P. aeruginosa PAO1 and E. coli IH3080 in medium B at concentrations up to 0.3 to 1%; data not shown). Synergistic bactericidal action. In the series of synergism experiments described above, we have measured the inhibition of bacterial growth which took place during the 18-h
incubation period. However, because rifampin is a bactericidal, rapidly acting antibiotic, it is possible to measure the HMP-induced sensitization to rifampin also by determining the viable counts of the target bacteria incubated for 2 h in the presence of HMP and rifampin. Approximately 10 ,ug of rifampin per ml was required to kill P. aeruginosa PAO1 during the incubation, whereas 30-fold less was as effective if the OM was simultaneously damaged by HMP (0.3%) (Fig. 6). Synergistic bacteriolysis. Detergents such as SDS and Triton X-100 are often rapidly lytic to target membranes and lyse instantaneously, for instance, gram-negative bacteria which have OMs damaged by EDTA, polymyxin B, or PMBN (10, 18, 22). Accordingly, we chose those detergents and developed a rapid microdilution lysis assay (see Materials and Methods) to see whether HMP has an immediate OM-damaging action. HMP did have such an action (Fig. 7). For instance, 85% lysis in 3 min was observed after the addition of 0.1% SDS plus 0.1% HMP to the bacterial suspension, whereas either 0.1% HMP alone (without SDS) or 0.1% SDS alone (without HMP) decreased the turbidity of the bacterial suspension by less than 10%. Essentially similar results were obtained by using Triton X-100 as the detergent. NPN uptake. NPN is an uncharged, very hydrophobic compound which, when excited in a hydrophobic milieu, has a characteristic bright fluorescence emission peak at 420 nm. That peak is lacking when NPN is in an aqueous milieu. As shown previously by several authors (5, 14, 17), NPN is a useful probe for detecting defective or damaged OMs which allow the penetration of NPN into hydrophobic membrane environments. Accordingly, we included NPN iT our set of probes and found that the fluorescence emission of NPN at 420 nm was significantly enhanced when the bacterial suspensions were treated with HMP (Table 1).
DISCUSSION
Gram-negative bacteria such as members of the family Enterobacteriaceae and especially Pseudomonas strains are
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HMP-INDUCED SENSITIZATION TO HYDROPHOBIC DRUGS
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remarkably resistant to hydrophobic noxious agents (10), and problems deriving from this property are often encountered in applied fields of microbiology. However, it seems to be possible to sensitize these bacteria to such agents. In this report, we have shown that HMP is an effective sensitizer. Thus, we found that HMP sensitizes the target bacteria effectively to all the various hydrophobic probe compounds included in our study. Furthermore, this sensitization was evident in all the various assay systems used. Thus, in the growth inhibition assay, 0.3% HMP decreased the MICs of the probes used against P. aeruginosa by a factor of approximately 10 (Fig. 1, 2, and 5), and maximally even a 30-fold sensitization was found with 1% HMP (Fig. 2C). In another assay, the bactericidal assay, 0.3% HMP decreased the MBC of the hydrophobic probe by a factor of approximately 30 (Fig. 6). Furthermore, HMP drastically and instantaneously sensitized the target bacteria to the lysis by detergents (Fig. 7) and increased the binding of our fluorescent hydrophobic probe (NPN) to their membranes (Table 1). Certain other metal chelators have previously been described to have a sensitizing action apparently analogous to that now found for HMP. Thus far, the best characterized in this respect is EDTA. Divalent cations (Mg2+ and/or Ca2+) are essential in linking anionic lipopolysaccharide molecules to each other in the OM, and by removing those divalent cations, EDTA causes a significant release of lipopolysaccharide from the OM (6, 8, 10). According to the current hypothesis (10), it is presumed that the empty space thus created is filled by phospholipids, which could explain the permeability to hydrophobic agents. Besides EDTA, another previously described sensitizing chelator is nitrilotriacetate (5). However, its sensitizing action has not yet been studied by any detail. It is very probable that the effect of HMP on the bacterial OM is analogous to that of EDTA. As shown in Fig. 3, 0.3% HMP appears to be as effective a sensitizer as is 3 mM
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VAARA AND JAAKKOLA
ANTIMICROB. AGENTS CHEMOTHER.
surprising, because P. cepacia has previously been shown to be resistant to EDTA (9). This suggests that the lipopolysaccharide molecules in the P. cepacia OM interact with each other and/or with the proteins without the aid of divalent cations. In analogy with these findings, P. cepacia is also resistant to polymyxins (9), which replace divalent cations in the polymyxin-sensitive lipopolysaccharides. The ability of HMP to sensitize gram-negative bacteria to hydrophobic compounds might have some value, for instance, in food microbiology. Polyphosphates are widely used in foods and food processing for emulsification, moisture retention, leavening, sequestering of cations, buffering, and improving tenderness (2, 3, 15). They are not usually regarded as antimicrobial agents or preservatives. On the other hand, several antimicrobial food additives or naturally occurring food antimicrobial agents are hydrophobic and therefore do not permeate effectively through the OM. Consequently, they are much less active against gramnegative bacteria than against gram-positive bacteria. By permeabilizing the OM, as shown in our report, HMP could be expected to sensitize the target bacteria to those antimicrobial agents. They include monoglycerides, sugar esters of fatty acids, free fatty acids, and thiocyanate and its metabolites (1, 2, 7, 12, 13). Through future research, it should be possible to further exploit the potential value of HMP as a part of such antimicrobial combinations. not
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determinations.
(approximately 0.1%) EDTA. The effect of HMP was found to be reversed by Mg2+ and pronounced in dilute growth media which have been described to be nearly limiting in terms of the supply of Mg2+ and Ca2+ (28). On the other hand, both HMP and EDTA were much weaker sensitizers than PMBN, the known very potent sensitizer which is not chelator but has a high affinity for bacterial lipopolysaccharides and is effectively bound to the OM (20-23, 27). Even as low a PMBN concentration as 1 pug/ml (equal to approximately 1 p,M) was more active than the high concentrations of EDTA and HMP tested (Fig. 3). As far as we know, there has been only one previous report which described any sensitizing or synergistic effect of HMP with hydrophobic drugs. That report (16) indicated that E. coli and Aeromonas hydrophila can be sensitized to glycerol monocaprate (100 p,g/ml), a commonly used preservative, by 0.5% HMP. No other concentrations of HMP or glycerol monocaprate were tested. Our present findings bring much more data about the sensitizing effect of HMP and widen the spectrum of susceptible bacteria to cover also P. aeruginosa and many other Pseudomonas strains. Of the Pseudomonas strains that were tested, only P. cepacia was resistant to the effects of HMP. This finding was TABLE 1. Effect of HMP on NPN fluorescence of P. aeruginosa cells HMP concn (%)
Increase in NPN fluorescencea
None 0.01 0.1 1
0 16.7 ± 9.5 43.7 ± 8.5 46.2 ± 3.0
a NPN fluorescence was measured 2 min after the addition of HMP to cells in 0.01 M HEPES (pH 7.4) with 50 mM NaCl and 10 ,uM NPN. Results are expressed in arbitrary units (mean + standard deviation of three independent experiments). Background fluorescence in the absence of HMP (12.6 ± 1.0) was subtracted.
ACKNOWLEDGMENT We thank Birgit Kuusela for her excellent technical assistance.
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39:1187-1195. 13. Shibasaki, I. 1982. Food preservation with nontraditional antimicrobial agents. J. Food Saf. 4:35-58. 14. Tecoma, E. S., and D. Wu. 1980. Membrane deenergization by colicin K affects fluorescence of exogenously added but not biosynthetically esterified parinaric acid probes in Escherichia coli. J. Bacteriol. 142:931-938. 15. Tompkin, R. B. 1983. Indirect antimicrobial effects in foods: phosphates. J. Food Saf. 6:13-27. 16. Tsutsumi, M., I. Suda, J. K. Lee, S. Kato, and T. Watanabe. 1983. Preservative effect by the combined use of polyphosphate, glycerol monocaprate and lysozyme. J. Food Hyg. Soc. Jpn. 24:301-307. 17. Uratani, Y. 1982. Dansyl chloride labeling of Pseudomonas aeruginosa treated with pyocin Rl: change in permeability of the cell envelope. J. Bacteriol. 149:523-528. 18. Vaara, M. 1981. Increased outer membrane resistance to ethylenediaminetetraacetate and cations in novel lipid A mutants. J. Bacteriol. 148:426-434. 19. Vaara, M. 1988. Analytical and preparative high-performance liquid chromatography of the papain-cleaved derivative of polymyxin B. J. Chromatogr. 441:423-430. 20. Vaara, M., and T. Vaara. 1983. Sensitization of gram-negative bacteria to antibiotics and complement by a nontoxic oligopeptide. Nature (London) 303:526-528.
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21. Vaara, M., and T. Vaara. 1983. Polycations sensitize enteric bacteria to antibiotics. Antimicrob. Agents Chemother. 24: 107-113. 22. Vaara, M., and T. Vaara. 1983. Polycations as outer membranedisorganizing agents. Antimicrob. Agents Chemother. 24:114122. 23. Vaara, M., and P. Viljanen. 1985. Binding of polymyxin B nonapeptide to gram-negative bacteria. Antimicrob. Agents Chemother. 27:548-554. 24. Van Wazer, J. R., and C. F. Callis. 1958. Metal complexing by phosphates. Chem. Rev. 58:1011-1046. 25. Van Wazer, J. R., and D. A. Campanelia. 1950. Structure and properties of the condensed phosphates. IV. Complex ion formation in polyphosphate solutions. J. Am. Chem. Soc. 72:655-663. 26. Viljanen, P., P. Koski, and M. Vaara. 1988. Effect of small cationic leukocyte peptides (defensins) on the permeability barrier of the outer membrane. Infect. Immun. 56:2324-2329. 27. Viljanen, P., and M. Vaara. 1984. Susceptibility of gramnegative bacteria to polymyxin B nonapeptide. Antimicrob. Agents Chemother. 27:548-554. 28. Wee, S., and B. J. Wilkinson. 1988. Increased outer membrane ornithine-containing lipid and lysozyme penetrability of Paracoccus denitrificans grown in a complex medium deficient in divalent cations. J. Bacteriol. 170:3283-3286.
