a Department of Gastrointestinal Infections, Statens Serum Institut, 2300 Copenhagen S, Denmark; b Department of Biochemistry, Microbiology and Molecular Genetics, University of Rhode Island, Kingston, RI 02881; c Legere Pharmaceuticals Inc., Carson City, NV 89706, USA
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Abstract |
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Introduction |
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A great deal of effort has gone into attempts to make the Gram-negative outer membrane permeable to antibiotics in the hope that this may prove useful clinically. Several polycations have been shown to make the outer membrane permeable, presumably by binding to lipopolysaccharide (LPS). These include polymixin B and its derivatives,9 including deacylpolymixin B10 and polymixin B nonapeptide.10 Other polycationic permeabilizers include bactericidal/permeability-increasing protein,11 protamine9 and various polycationic peptides,1216 including lysine polymers, defensins, cecropins, magainins and mellitin.
Chelators of divalent cations, such as ethylenediaminetetraacetate (EDTA), nitrilotriacetate and sodium hexa-metaphosphate, are all effective in making outer membranes permeable to antibiotics (reviewed in references 1719). Chelators presumably render the membrane permeable by removing Ca2+ and Mg2+ from LPS, resulting in release of much of the LPS from the outer membrane and consequent outer membrane destabilization (reviewed in references 1719). Phospholipids are also known to bind Ca2+ and Mg2+ effectively,20 suggesting that certain phospholipids might make the outer membrane of P. aeruginosa permeable to ß-lactam antibiotics. This hypothesis is examined in the present study.
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Materials and methods |
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P. aeruginosa PAO121 and P. aeruginosa AC869, a strain that degrades 3,5-dichlorobenzoate,22 were obtained from Dr S. E. George of the National Health and Environmental Effects Research Laboratory, USEPA, Research Triangle Park, NC, USA. P. aeruginosa H103, another prototrophic PAO1 strain,23 and H636, a protein F-deficient strain derived from H103,24 were obtained from Dr R. E. W. Hancock, Department of Microbiology, University of British Columbia, Vancouver, British Columbia, Canada.
P. aeruginosa strains 17 AM, 47 AL, 77AM and 82 AM, mucoid strains isolated from the sputum of individual chronically infected cystic fibrosis (CF) patients, were obtained from Dr G. Pier, Channing Laboratory, Harvard Medical School, Boston, MA, USA. P. aeruginosa strains PAO 579, 17107 and 19676 were obtained from Dr Niels Høiby, Danish Cystic Fibrosis Centre, Department of Clinical Microbiology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.
Laboratory media and antibiotics
Strains were routinely grown at 37°C in Luria broth (L-broth) and plated on L-agar (L-broth containing 12% (w/v) Bacto-agar [Difco, Detroit, MI, USA)]. L-broth was prepared as described by Revel.25 Ampicillin was purchased from Sigma, St Louis, MO, USA.
Phospholipids
All phospholipids were purchased from Avanti Polar-Lipids, Inc., Alabaster, AL, USA. Phospholipids were dispersed in L-broth as described in the text.
Measuring Ca2+ and Mg2+ in L-broth
Ca2+ concentrations were determined using a Beckman Synchron EL-ISE Electrolyte System (Beckman Coulter Inc., Fullerton, CA, USA). This electrode simultaneously measures Ca2+, Na+, K+ and Cl concentrations. Mg2+ concentrations were determined using a Beckman Synchron CX-7 Autoanalyzer (Beckman Coulter Inc.).
MIC and MBC of ampicillin
In this study, the MIC was defined as the lowest concentration of ampicillin that reduced growth by more than one order of magnitude relative to the untreated culture and the MBC as the lowest concentration of ampicillin that resulted in a cfu/mL of an order of magnitude lower than that at inoculation.
Determination of MPPA concentrations in MPPA supernatants
Monopalmitoylphosphatidic acid (MPPA) is the lysophospholipid 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate. MPPA supernatants (30 µL) and L-broth with no added MPPA (30 µL) were dissolved in 0.45 mL of 8.9 N H2SO4 and heated at 210°C for 25 min. Samples were allowed to cool for 5 min, then six drops of hydrogen peroxide were added to each sample and samples were heated at 210°C for 30 min. Inorganic phosphate released by this hydrolysis procedure was determined as described by Chen et al.26 In control experiments, essentially 100% of the inorganic phosphate in known amounts of MPPA was detected by this method. The concentration of inorganic phosphate in L-broth was subtracted from the total concentration of inorganic phosphate in each MPPA supernatant to yield the concentration of MPPA inorganic phosphate. All samples were run in triplicate. The concentration of MPPA in any supernatant was calculated based on the concentration of MPPA inorganic phosphate in the supernatant and the percentage (by weight) of inorganic phosphate in MPPA.
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Results |
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The MIC of ampicillin for P. aeruginosa PAO1 in L-broth is 2 g/L. A suspension of dipalmitoylphosphatidylserine (DPPS) in L-broth was prepared by sonication for 5 min in an ultrasonic water bath at room temperature and was inoculated with P. aeruginosa PAO1 (5 x 104 cfu/mL). A second suspension of DPPS in L-broth containing ampicillin 200 mg/L was also inoculated with P. aeruginosa PAO1 in the same way. As controls, L-broth containing neither ampicillin nor DPPS and L-broth containing ampicillin 200 mg/L were inoculated with P. aeruginosa PAO1. Cultures were incubated with aeration for 18 h at 37°C before plating. P. aeruginosa PAO1 reached concentrations of about 1010 cfu/mL in L-broth, in L-broth containing DPPS in suspension and in L-broth containing ampicillin. In contrast, it did not grow in L-broth containing both DPPS 1 g/L in suspension and ampicillin 200 mg/L. Thus, DPPS in suspension appeared to enhance ampicillin activity against P. aeruginosa PAO1.
DPPS supernatants enhance ampicillin activity against P. aeruginosa PAO1
DPPS 1 g/L was sonicated into L-broth for 5 min in an ultrasonic water bath at room temperature and then incubated for 4 h at 37°C in a circulating water bath. Undissolved DPPS was removed by centrifugation at 12000g for 10 min at 4°C. The supernatant (preincubated DPPS supernatant, or P-SN) was used as the source of dissolved DPPS. As a control, DPPS 1 g/L was sonicated into L-broth for 5 min and undissolved DPPS was immediately removed by centrifugation as described above. The supernatant is called the non-preincubated DPPS supernatant (NP-SN). Aliquots (1 mL) of L-broth, NP-SN and P-SN containing either no ampicillin or ampicillin at 500, 200 or 50 mg/L were inoculated with 5.0 x 103 cfu/mL of P. aeruginosa PAO1. Cultures were incubated with aeration for 18 h at 37°C, at which time cultures were plated for viable counts.
Viable counts showed that neither NP-SN nor P-SN prevented P. aeruginosa PAO1 growth in the absence of ampicillin (Table I). Although NP-SN was effective in preventing P. aeruginosa PAO1 growth in the presence of ampicillin 500 mg/L, it was less effective with ampicillin 200 mg/L and was ineffective with ampicillin 50 mg/L (Table I
). In contrast, P-SN completely prevented P. aeruginosa PAO1 growth at ampicillin 50 mg/L (Table I
). P-SN was clearly superior to NP-SN in enhancing the activity of ampicillin against P. aeruginosa PAO1, suggesting that DPPS disperses slowly in L-broth, and that DPPS supernatants can be prepared to enhance ampicillin activity against P. aeruginosa PAO1.
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The following components of DPPS were tested for their ability to enhance the activity of ampicillin 200 mg/L against P. aeruginosa PAO1: glycerol, glycerol 3-phosphate, dipalmitoylglycerol, glycerophosphorylserine, phosphorylserine, palmitic acid and dipalmitoylphosphatidic acid (DPPA). Each of these compounds was tested at concentrations present in DPPS at 1 g/L. The components were individually sonicated into L-broth for 5 min in an ultrasonic water bath at room temperature and then incubated for 4 h at 37°C in a circulating water bath. Any undissolved material was removed by centrifugation at 12000g for 10 min at 4°C and the supernatants were used as the source of dissolved compounds. Cultures (1 mL) containing either no ampicillin or ampicillin 200 mg/L were inoculated with P. aeruginosa PAO1, incubated and plated as described above. As shown in Table II, only DPPA had ampicillin-enhancing activity comparable to that of DPPS. The fact that dipalmitoylglycerol and glycerophosphorylserine did not enhance the activity of ampicillin shows the importance of the phosphate and fatty acids, respectively. In support of this view, the only other compound that had any enhancing activity, although weak, was palmitic acid (Table II
).
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Supernatants made from several other phospholipids were tested for their ampicillin-enhancing activity. Each of the supernatants was made based on their molecular weights relative to DPPS. The phospholipids tested were: MPPA, which contains one palmitic acid (C16) on C1 of the glycerol backbone; monomyristoylphosphatidic acid (MMPA), which contains one myristic acid (C14) on C1 of the glycerol backbone; dilauroylphosphatidic acid (DLPA), which contains one lauric acid (C12) on C1 and one lauric acid on C2 of the glycerol backbone; dicaproylphosphatidic acid (DCPA), which contains one caproic acid (C6) on C1 and one caproic acid on C2 of the glycerol backbone; dipalmitoylphosphatidylcholine (DPPC), which contains a palmitic acid on both C1 and C2 of the glycerol backbone and phosphorylcholine on C3 of the glycerol backbone; and monopalmitoylphosphatidylcholine (MPPC), which is like DPPC but without the palmitic acid on C2 of the glycerol backbone. The ampicillin-enhancing activities were in the order MPPA and MMPA > DPPA > DPPS = DLPA. DCPA, DPPC and MPPC had no enhancing activity (Table III).
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Supernatants (preincubated for 24 h at 37°C), made using 100 mg/L of MPPA and MMPA rather than the higher concentrations normally used, were tested for the ability to enhance the activity of ampicillin 200 mg/L. Under these conditions, the cell density of P. aeruginosa PAO1, in cfu/mL, increased only eight-fold (compared with the inoculum) in 18 h at 37°C in the presence of MPPA, but 14000-fold in the presence of MMPA (not shown). Since MPPA was the better enhancer of ampicillin activity, it was used in all further experiments.
MPPA enhancement of ampicillin activity using different inocula
Preincubated (4 h at 37°C) supernatant, made using 570 mg/L of MPPA in L-broth, was inoculated with between 5 x 103 and 5 x 107 cfu/mL of P. aeruginosa PAO1 in the presence of ampicillin 200 mg/L and incubated for 18 h at 37°C before plating. The preincubated MPPA supernatant was extremely effective in enhancing the activity of ampicillin against an inoculum of 5.3 x 106 cfu/mL and even at an inoculum of 5.3 x 107 cfu/mL, 97.5% of the inoculum was killed in the presence of ampicillin (Table IV).
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The non-ionic detergent Brij-58 was used in an attempt to increase the solubility of DPPS and MPPA. DPPS 1 g/L was sonicated into L-broth containing 1% Brij-58, inoculated with 4 x 103 cfu/mL of P. aeruginosa PAO1 in the absence or presence of ampicillin 500 mg/L and incubated for 18 h at 37°C before plating. Surprisingly, Brij-58 reversed the ampicillin-enhancing activity of DPPS, resulting in 1.4 x 109 cfu/mL of PAO1 in the presence of ampicillin 500 mg/L. Similarly, Brij-58 reduced the enhancing activity of a 570 mg/L MPPA supernatant. Essentially the same results were observed in the presence of 0.1% Triton X-100, another non-ionic detergent (data not shown).
MIC and MBC of ampicillin in the presence of MPPA
Preincubated supernatant (4 h at 37°C), made using 570 mg/L of MPPA in L-broth and containing ampicillin at concentrations of 10, 20, 40, 60, 80, 100 and 200 mg/L, was inoculated with P. aeruginosa PAO1 (3.5 x 103 cfu/mL) and the MIC and MBC were determined. The MBC of ampicillin, as judged by killing of the inoculum (one order of magnitude), was 40 mg/L and the MIC, as determined by significant inhibition of growth (one order of magnitude), 20 mg/L.
Reversal of MPPA enhancement of ampicillin activity by Ca2+ and Mg2+
EDTA makes the Gram-negative outer membrane permeable to antibiotics by chelating Ca2+ and Mg2+ and thereby destabilizing the membrane.1719 Phospholipids are also known to have an affinity for Ca2+ and Mg2+.20 Therefore, we performed experiments in an attempt to determine whether MPPA might be binding Ca2+ and Mg2+ and thereby rendering PAO1 sensitive to ampicillin. First, calcium chloride (final concentrations of 200 and 400 µM) was added to MPPA 570 mg/L preincubated supernatants. Whereas MPPA still enhanced ampicillin activity against P. aeruginosa PAO1 when the Ca2+ concentration was increased by 200 µM, MPPA enhancement of ampicillin activity was lost when the Ca2+ concentration was increased by 400 µM. Second, the Ca2+ concentration in L-broth was increased by 400 µM, MPPA was added to a concentration of 2 g/L, the mixture was incubated at 37°C for 4 h and then centrifuged to remove any undissolved MPPA. MPPA supernatants made in this way were effective in enhancing the activity of ampicillin. Third, MPPA was added to a concentration of 2 g/L in L-broth, the mixture was incubated at 37°C for 4 h, undissolved MPPA was removed by centrifugation and then Ca2+ was added to a final concentration of 400 µM. Upon addition of the Ca2+, a visible white precipitate appeared, suggesting that a Ca2+phospholipid complex had formed. Under these conditions, ampicillin activity against P. aeruginosa was still enhanced (data not shown).
Addition of Mg2+ did not reverse MPPA enhancement of ampicillin activity to the same extent as addition of Ca2+. P. aeruginosa was still killed in the presence of MPPA, ampicillin and an additional 200 µM Mg2+ and was not killed, but did not grow, in the presence of MPPA, ampicillin and an additional 400 µM Mg2+ (data not shown).
MPPA binds Ca2+ and Mg2+
The Ca2+ and Mg2+ concentrations in L-broth were 263 and 225 µM, respectively (see Materials and methods). MPPA supernatants preincubated for 4 h were made by adding 100 mg/L of MPPA to L-broth and removing undissolved MPPA by centrifugation. Under these conditions, the Ca2+ and Mg2+ concentrations were 73 and 200 µM, respectively; Ca2+ was thus preferentially removed by MPPA. However, when MPPA supernatants (570 mg/L) were examined after centrifuging out undissolved MPPA, the Ca2+ and Mg2+ concentrations were 73 and 29 µM, respectively. When MPPA supernatants (2 g/L) were examined after centrifuging out undissolved MPPA, the Ca2+ and Mg2+ concentrations were 55 and 13 µM, respectively. These data show that MPPA binds both Ca2+ and Mg2+ and, in doing so, forms a precipitate. Na+, K+ and Cl concentrations remained constant under these conditions at 103, 7.5 and 94 mM, respectively.
Determination of MPPA concentrations in MPPA supernatants
Three independently prepared supernatants made from MPPA suspensions in L-broth at concentrations of 570 mg/L, 1 g/L and 2 g/L were assayed for phospholipid phosphate as described in Materials and methods. The concentrations of MPPA in each of the supernatants after centrifuging out undissolved MPPA were as follows: 570 mg/L added, 85 ± 7.8 mg/L in suspension; 1 g/L added, 135 ± 27 mg/L in suspension; 2 g/L added, 422 ± 38 mg/L in suspension.
Screening of environmental and CF P. aeruginosa strains for MPPA-enhanced sensitivity to ampicillin
P. aeruginosa AC869,22 H103 and H636, a protein F-deficient mutant of H10323 (protein F is thought to be involved in transport of ampicillin24) and P. aeruginosa PAO579, a non-mucoid strain isolated from a CF patient (see Materials and methods) were tested for MPPA-enhanced sensitivity to ampicillin. All four strains became sensitive to ampicillin in the presence of MPPA (Table V). The data showing that H103 and H636 both become sensitive to ampicillin in the presence of MPPA rule out the possibility that protein F transports ampicillin better in the presence of MPPA.
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Ceftazidime, a third-generation cephalosporin, is used to treat CF patients chronically infected with P. aeruginosa.27 One moderately ceftazidime-resistant isolate, number 19676 (MIC 10 mg/L) and one highly ceftazidime-resistant isolate, number 17107 (MIC 100 mg/L), both isolated from chronically infected CF patients, were treated with ceftazidime in the presence or absence of MPPA. In both cases, MPPA enhanced the activity of ceftazidime (Table VII).
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Gentamicin is an aminoglycoside inhibitor of protein synthesis and nalidixic acid is a quinolone inhibitor of DNA gyrase. The MICs of gentamicin and nalidixic acid for strain PAO1 were 1.56 and 25 mg/L, respectively. Preincubated (4 h at 37°C) supernatant, made using 570 mg/L of MPPA in L-broth, was inoculated with 1.0 x 104 cfu/mL of P. aeruginosa PAO1 in the presence of ampicillin 200 mg/L, gentamicin 0.4 mg/L or nalidixic acid 6.25 mg/L and incubated for 18 h at 37°C before plating. The MPPA supernatant had no enhancing effect on gentamicin or nalidixic acid at these concentrations, while it was effective in enhancing the activity of ampicillin (data not shown).
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Discussion |
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Since DPPA is an excellent enhancer of ampicillin activity against P. aeruginosa PAO1, while glycerol 3-phosphate and dipalmitoylglyerol are not (Table II), it appears that both the phosphate and fatty acid moieties are important. The length of the fatty acid moieties also appears to be important: dilauroylphosphatidic acid (C12) is a good enhancer of ampicillin activity, whereas dicaproylphosphatidic acid (C6) has no enhancing activity.
Three lines of evidence suggest that, for a phospholipid to enhance ampicillin activity against P. aeruginosa, it must be able to form lipid bilayers or micelles, both of which contain high negative charge density at the surface.20 (i) Both the phosphate and fatty acid moieties of the phospholipid molecules are important for enhancement (Table II) and these moieties are required for both micelle and lipid bilayer formation.20 (ii) Dicaproylphosphatidic acid is freely soluble in L-broth, but is unable to form lipid bilayers or micelles20 and has no ampicillin-enhancing activity (Table III
). (iii) Increasing the solubility of the phospholipids with Brij 58 or Triton X-100 diminished its enhancing activity, presumably by decreasing micelle formation.
Liposome-encapsulated ß-lactam antibiotics are more effective against P. aeruginosa than the antibiotic itself, both in vivo and in vitro.28,29 The mechanism by which this occurs is not clear. From the present investigation, it appears that the mechanism by which MPPA enhances ß-lactam antibiotic activity against P. aeruginosa is similar to the mechanism by which EDTA, a metal chelator, sensitizes P. aeruginosa to a number of antibiotics, including ß-lactams.30,31 EDTA is thought to chelate Ca2+ and Mg2+ from the LPS of the P. aeruginosa outer membrane, causing release of LPS, thereby destabilizing the membrane and making it permeable to a variety of antibiotics (reviewed in references 1719). In the present study, we have shown that MPPA binds both Mg2+ and Ca2+ in L-broth and precipitates, thereby reducing the Ca2+ and Mg2+ concentrations as much as four- to five-fold. Reduction in Ca2+ may be more important than reduction in Mg2+ since addition of 400 µM Ca2+ to MPPA supernatants (570 mg/L) completely reversed the enhancement of ampicillin activity whereas addition of 400 µM Mg2+ was not as effective.
The enhancement of antibiotic activity by MPPA may be limited to the ß-lactam antibiotics, as the activities of the aminoglycoside gentamicin and the quinolone nalidixic acid were not enhanced by MPPA. Further investigation will be required to determine whether the activities of any classes of antibiotics other than the ß-lactams are enhanced by MPPA.
We have shown that MPPA activity is not restricted to P. aeruginosa PAO1. Ten additional P. aeruginosa strains that grow in the presence of MPPA were tested for sensitivity to ampicillin, piperacillin or ceftazidime in the presence of MPPA. All 10 became sensitive (Tables VVII). Surprisingly, MPPA by itself slowed the overnight growth of four different mucoid antibiotic-resistant strains of P. aeruginosa isolated from the sputum of chronically infected CF patients (Table VI
). This finding suggests that the exopolysaccharide alginate does not interfere with the action of MPPA, which is significant, since mucoid strains produce copious quantities of alginate in vivo.32,33 Furthermore, unlike environmental strains and strains isolated during the initial stages of infection of CF patients, the majority of P. aeruginosa strains isolated from chronically infected CF patients contain little, if any, O-antigen on their LPS.34,35 LPS mutants are known to be more sensitive to detergents than their wild-type parents.18,19 Therefore it would be of great interest to determine whether the reduced O-antigen on these strains is responsible for the ability of MPPA by itself to slow their growth and whether MPPA or some other phospholipid by itself will be effective in treating pulmonary P. aeruginosa infections in chronically infected CF patients. In addition, it would be of interest to determine whether MPPA or some other phospholipid, in concert with appropriate antibiotics, will be useful in more effectively treating burn patients and patients with eye infections caused by P. aeruginosa.
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Acknowledgments |
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Notes |
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References |
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Received 6 August 1999; returned 4 November 1999; revised 20 March 1000; accepted 10 April 2000