Journal of Applied Bacteriology 1992,73, 484-408

Effect of polysaccharide interactions on antibiotic susceptibility of Pseudomonas aeruginosa D.G. Allison and M.J. Matthews Pharmacy Department, Manchester University, Manchester, UK 4177103192:accepted 26 June 1992 D.G. A L L I S O N A N D M.J. MATTHEWS. 1992.T h e relative viscosity of Pseudomonas aeruginosa alginate was shown to increase markedly when combined with mucin, C a 2 + ions and the exopolysaccharide from Pseudomonas cepacia. T h e presence of such a heterodisperse polysaccharide solution significantly reduced the diffusion and hence antimicrobial activity of tobramycin and to a lesser extent ciprofloxacin against Ps. aeruginosa by factors of 90 and 2-5-fold respectively over a 5 h incubation period. T h e clinical implications of these results are discussed in relation t o cystic fibrosis.

INTRODUCTION

Pseudomonas aeruginosa infections continue to represent a major threat to patients with fibrocystic (CF) lung disease. Once acquired, such populations are seldom, if ever, eliminated from the lung by conventional antibiotic therapy, resulting eventually in death through pulmonary failure. Although the initial infecting strains of Ps. aeruginosa are non-mucoid, with continued proliferation in the lung, they become highly mucoid due to the production of an alginate-like exopolysaccharide (EPS). It has been suggested that expression of the mucoid phenotype may afford protection to the underlying cells by retarding antibiotic diffusion (Gordon et al. 1988; Nicholls et al. 1989). The rheological properties of alginate are influenced by the molecular weight and polyanionic nature of the EPS. As such, alginate gelation is induced by the presence of divalent cations, particularly calcium, which has been shown to decrease the rate of aminoglycoside diffusion through such gels (Gordon et al. 1988). The clinical relevance of these studies is limited, however, by the lack of data concerning rheological interactions between different polysaccharides present in the lung. Characteristic to C F is the overproduction of an abnormally viscous mucous (Goodchild 8i Dodge 1985). Furthermore, Ps. cepacia, which also has the ability to produce large amounts of EPS (Sage et al. 1990), can co-inhabit the lung with Ps. aeruginosa (Gilligan 1991). Hence, the purpose of the present study was to determine the nature of EPS interactions occurring between different bacterial and host polysaccharides and to assess the effect upon antibiotic penetration. Correspondence to Dr D.G. Allison, Pharmacy Department, Manchcsicr University, Oxford Road, Manchestcr MI3 9PL, UK.

MATERIALS AND METHODS Bacteria and culture conditions

Pseudomonas aeruginosa strain PaWH (mucoid), isolated from the sputum of a CF patient and Ps. cepacia NCTC 10661 were grown at 37°C on yeast extract (YE) media as previously described (Allison & Sutherland 1987). Stock cultures were maintained at -20°C in nutrient broth containing 10% glycerol. Exopoiysaccharide extraction and purlflcatlon

Exopolysaccharide was isolated from cell suspensions according to the methods described by Allison & Sutherland (1987). Lyophilized EPS was purified by re-dissolving (25 mg/ml) in 0.01 mol/l ammonium bicarbonate buffer (pH 8.5) and fractionating on an equilibrated DEAE cellulose column (2.6 cm x 30 cm). Elution with a linear gradient of 0.01-1.0 moll ammonium bicarbonate at 50 ml/h permitted fractions (2.5 ml) to be collected and assayed for total carbohydrate (Dubois et al. 1956). Carbohydratecontaining fractions were pooled, concentrated and further fractionated by gel-filtration with Sephacryl S-500 and 0.5 mol/l Tris-HCI, 0-15 mol/l NaCl eluent (pH 8.8, 30 ml/h). Viscosity determinations

Exopolysaccharide viscosity was determined for : (i) purified polymers of Ps. aeruginosa, Ps. cepacia and partially purified commercially-available mucin (Sigma) at various concentrations ; (ii) Ps. aeruginosa (various concentrations) in the presence of cations (1% w/v) ; and (iii) a combination of all three polymers at equal concentrations plus calcium ions (1% w/v) using a simple U-tube capillary viscometer

EX 0P 0 L Y S A C C H A R I DES A N D ANT I B I0T I C ACT I V I TY 485

designed for small volume samples. The time taken (s) for the sample to fall a fixed distance under gravity at 35°C was taken as a measure of relative viscosity, Measurements were repeated in triplicate to an accuracy of 0.5 s. Antibiotic susceptibility testing

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Preliminary experiments, utilizing mid-log batch culture of the test organism, were conducted to establish those concentrations of ciprofloxacin (0.5 pg/ml) and tobramycin (20 pg/ml) which gave appropriate levels of killing (1-2 log cycles) within a 1 h contact period at 35°C. Levels of survival in all cases decreased with increasing drug concentration. These concentrations were subsequently used to assess the effect of EPS rheological interactions on antibiotic penetration. Appropriate controls were incorporated in each experiment to circumvent antibiotic/ionic effects. T o assess the rate of killing, a dialysis bag (6 kDa cut off) containing either antibiotic (1 ml) at the above concentrations or antibiotic plus 1% (w/v) EPS solution (1 ml) was aseptically added to a 250 ml Erlenmeyer flask containing 100 ml Ps. aeruginosa cell suspension resuspended to a concentration of 5 x lo7 cells/ml in YE salts (Allison & Sutherland 1987). Samples were removed at regular intervals, serially diluted in sterile normal saline and viable counts made on the surfaces of pre-dried nutrient agar plates in triplicate. All plates were subsequently incubated at 35°C for 16 h. Results were expressed as percentage reductions in viability relative to appropriate unexposed controls.

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Flg. 1 EffPct of cations on Pseudomonas aeruginosa PaWH alginate viscosity. Control, no added ions ( 0 ) Na+ ; (0); A13+ (H), Mg2+ ( 0 )and Ca2+ (A)

alginate and the EPS obtained from Ps. cepacia possessed similar rheological properties, increasing in viscosity with polysaccharide concentration. Mucin on the other hand, showed a different response. Whilst a low viscosity similar

RESULTS Vlscoslty measurements

The effect of different cations on the viscosity of Ps. aemginosa PaWH alginate is shown in Fig. 1. Addition of Na+ ions had little effect on the overall viscosity of the polymer, particularly at low polysaccharide concentrations. A slight increase in polymer gelation was observed with the addition of Mg2+ and A13+ ions. Both cations demonstrated a similar increase in polymer viscosity irrespective of polysaccharide concentration, relative to the control solution in the absence of ions. In contrast, addition of Ca2+ demonstrated a polysaccharide concentrationdependent effect. At low alginate concentrations ( 0*2%), a marked increase in polysaccharide viscosity was observed. As such, changes in PaWH alginate viscosity were concentration- and cationdependent. Relative viscosity measurements for the different polysaccharides are shown in Fig. 2. I t is evident that both the

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Fig. 2 Relative viscosity of Pseudomonas aeruginosa PaWH alginate,).( Ps.cepaciu EPS (O), Mucin (H),all three polymers ( 0 )and all three polymers + 1% (w/v) Caz+ (A)

486 D.G. ALLISON AND M.J. MATTHEWS

to that of alginate and Ps. cepacia EPS was measured for a 0.1 % solution, increasing the concentration of mucin resulted in a greater increase in viscosity than observed for alginate and Ps. cepacia EPS. Such a response implies complex interactions occurring within the mucin polymer as the concentration increases. When all three polymers were combined in equal amounts, a marked increase in relative viscosity was observed. This effect was dramatically heightened by the addition of 3 mmol/l calcium ions to the combined polymeric solution. T h e same Ca2 induced effect was not observed with any one individual polymer but was dependent upon the presence of alginate, mucin and Ps. cepacia EPS. +

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Figures 3 and 4 illustrate the effect of polysaccharide interactions on antibacterial properties of tobramycin and ciprofloxacin respectively, against Ps. aeruginosa PaWH. After 5 h incubation in the presence of tobramycin (Fig. 3), ca 99.5% of the control population were killed. Addition of a 1% alginate solution to the antibiotic resulted in a significant increase in the number of survivors after a similar

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Fig. 4 Influence of polysaccharide solution (1 % W/V final

concentration) on ciprofloxacin activity against Pseudomonas aeruginosa. Control, no polysaccharide (a).All other samples contained ciprofloxacin plus Ps. aeruginosa PaWH alginate (O), Ps. aeruginosu PaWH alginate + mucin (H),Ps. aeruginosa PaWH alginate + mucin + Ca2+ (01, Ps. aeruginosa P a m alginate + much + Caz++ Ps. cepacia EPS (A)

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period of incubation to 60%. Whilst the addition of mucin did not cause a notable increase in the number of survivors, inclusion of Ca2 ions increased the population surviving to 75%. This was further increased to ca 90% by the addition of Ps. cepacia EPS. Indeed, this level of killing was reached after 3 h incubation and did not decrease thereafter. Surprisingly, ciprofloxacin activity was also affected by the polysaccharide solutions (Fig. 4). At a concentration of 0.5 pg/ml, ca 81% of the control population were killed after 5 h incubation. Addition of alginate and mucin had litde effect, the surviving population remaining at 21 %. Although Ca2+ ion gelation did cause an increase in survival to 30%, the most significant effect occurred with the incorporation of Ps. cepacia EPS to the polysaccharide solu\ . & & I tion. Under these conditions, 50% of the population sur3 4 5 6 vived the ciprofloxacin treatment after 5 h incubation. +

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Fig. 3 Influence of polysaccharide solution (1 % w/v final concentration)on tobramycin activity against Pseudomonas aeruginosu. Control, no polysaccharide (a).All other samples contained tobramycin plus Ps. aeruginosa PaWH alginate (O),Ps. aeruginosu PaWH alginate + mucin (m), Ps. aeruginosa PaWH alginate + mucin + Ca2+ (o),Ps. aeruginosa PaWH alginate + mucin + Ca2+ + Ps. cepucia EPS (A)

DISCUSSION

Bacteria interact with their own environment through the cell envelope. Consequently, a high degree of flexibility in structure and composition is shown in response to changes in the growth environment (Ellwood & Tempest 1972; Brown & Williams 1985a). Indeed, it is well documented

EXOPOLYSACCHARIDES AND ANTIBIOTIC ACTIVITY

that changes associated with growth rate and nutrient deprivation can profoundly influence antimicrobial susceptibility (Brown & Williams 1985b; Gilbert et al. 1990). Moreover, growth as a biofilm can also affect cell properties (Brown et al. 1988).Adherent micro-organisms differ from planktonic cells with respect to many important surface features (Allison et al. 1990, 1991) and can adopt phenotypes, characteristic to the growth environment, which can be particularly recalcitrant to antimicrobial agents (Brown et al. 1988; Gilbert et al. 1990). However, although environmental modulation contributes to the pathophysiology of chronic infections such as CF, there are other important factors that equally merit consideration. One such feature in CF is the rheological interaction between host and bacterial secretions. Clinically, patients suffering from C F accumulate in their lung highly dehydrated, viscous mucins with considerable amounts of electrolytes. Ultimately, such conditions lead to infections by Ps. aeruginosa which are found as slow growing, iron-limited (Anwar et al. 1984) biofilm populations (Costerton et al. 1987), enmeshed within EPS containing matrices (Lam et al. 1980). In addition, mucoid Ps. cepacia has recently emerged as a potentially important pathogen in C F (Gilligan 1991;Sage et al. 1990). Hence, in the fibrocystic lung microenvironment there is a complexity of different bacterial and host polysaccharides. An important property of many microbial polysaccharides is their ability to form gels, particularly in the presence of ions. Alginates form gels by the selective cooperative binding of Ca2+ ions, the magnitude of which is determined by the proportion of polyguluronic acid sequences (Sutherland 1990). Moreover, the interaction of alginate with other polysaccharides is particularly sensitive to Ca2+ concentration (Russel & Gacesa 1988). In CF patients, the level of Caz+ has been reported to be in the order of 3 mmol/l (Kilbourn 1984), and as such, is high enough to impact on alginate-based interactions. The results presented in this study clearly demonstrate the gelation effect caused by the formation of a heterodisperse polysaccharide solution in the presence of ions. Although the influence of Ca2+ was dependent on alginate concentration (Fig. l), in the combined presence of much and Ps. cepacia EPS there was a dramatic increase in viscosity (Fig. 2). The occurrence of this situation in vivo will exacerbate the problem of treating and clearing mucoid Ps. aeruginosa microcolonies which interact with mucosal surfaces in the CF lung (Costerton et al. 1987). Antibiotic resistance in CF infections has been largely attributed to failure to penetrate the EPS matrix associated with the biofilm (Gordon et al. 1988, 1991; Nicholls et al. 1989).This is particularly true for the aminoglycosides, and to a limited extent, B-lactam antibiotics (Gordon et al. 1988).Such studies, however, have either measured rates of

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antibiotic diffusion (Gordon et al. 1988) or predicted antibiotic equilibration times across homodisperse EPS slime layers (Nicholls et al. 1989). T o our knowledge there has been no direct assessment of antibiotic activity per se, nor any consideration on the influence of a heterodisperse polysaccharide solution on activity. Clearly, the presence of alginate reduces the rate of tobramycin diffusion. As a consequence, the number of cells killed by tobramycin over a given period of time is also reduced (Fig. 3). Indeed, these results confirm previous studies demonstrating tobramycin binding to bacterial alginate (Gordon et al. 1988). The effect of tobramycin on cell viability is further reduced both by the addition of CaZ+ ions, and more so by the inclusion of Ps. cepacia EPS (Fig. 3). Reduction in tobramycin activity can be explained on the basis of noncovalent associations occurring between the different polysaccharides, forming a three-dimensional 'weak-gel' network, cross-linked by Caz+ions. In this manner tobramycin is bound to the negatively-charged polysaccharide gel and prevented from reaching the cells. What is less clear is the underlying reason behind the reduced diffusion of ciprofloxacin through the polymeric solutions (Fig. 4). Since ciprofloxacin is uncharged, it is possible that the polymeric gel is implicated in non-diffusion-related resistance to the Cquinolones. Such a result may offer an explanation as to why ciprofloxacin has been effective in the therapy of other microbial chest infections, yet is unable to eradicate either Ps. aeruginosa or Ps. cepacia from the lungs of CF patients (Fass 1987). The clinical assessment of such studies is complicated since alginates isolated from the CF lung show considerable compositional heterogeneity (Pederson et al. 1989).There is also little information detailing the concentration of EPS and size of the microcolonies in vivo. Similarly, the ability of Ps. cepacia to synthesize EPS has only recently been recognized. Thus, although studies utilizing homodisperse polysaccharide solutions have their value (Gordon et al. 1991), they do not reflect the rheological interactions occurring in vivo within heterodisperse polysaccharide solutions. Such polysaccharide gels are more representative of those found in the CF lung and merit further investigation in order to improve antibiotic penetration characteristics. ACKNOWLEDGEMENT

This work was supported in part by a bursary from the Nuffield Foundation to DGA. REFERENCES A L L I S O N , D . G . & S U T H E R L A N D I, . W . (1987) The role of exopolysaccharides in adhesion of freshwater bacteria. Journal of General Microbiology 133, 1319-1327.

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