INFECTION AND IMMUNITY, May 1990, p. 1344-1349 0019-9567/90/051344-06$02.00/0 Copyright C) 1990, American Society for Microbiology

Vol. 58, No. 5

Oxygen-Dependent Up-Regulation of Mucoid Exopolysaccharide (Alginate) Production in Pseudomonas aeruginosa ARNOLD S. BAYER,l.2* FERESHTEH EFTEKHAR,3 JEREMY TU,1 CYNTHIA C. NAST,2'4 AND DAVID P. SPEERT3 Departments of Medicine' and Pathology,4 Harbor-UCLA Medical Center, Torrance, California 90509; University of

California at Los Angeles School of Medicine, Los Angeles, California 900242; and Department of Pediatrics, University of British Columbia, Vancouver, British Columbia VSZ 4H4, Canada3 Received 21 November 1989/Accepted 10 February 1990

We previously showed substantial differences in Pseudomonas aeruginosa exopolysaccharide production in vitro at oxygen tensions reflective of the right versus left cardiac circuits in vivo (40 versus 80 mm Hg, respectively; A. S. Bayer, T. O'Brien, D. C. Norman, and C. C. Nast, J. Antimicrob. Chemother. 23:21-35, 1989). However, those studies did not specifically confirm this exopolysaccharide to be the characteristic P. aeruginosa mucoid alginate seen in patients with cystic fibrosis. With a murine monoclonal antibody prepared against P. aeruginosa alginate, strongly positive immunofluorescence (IF) staining of a nonmucoid P. aeruginosa strain (PA-96) was seen after its exposure in vitro to oxygen tensions (PO2) of -80 mm Hg; the intensity of the IF staining under these conditions was similar to that observed with a phenotypically mucoid P. aeruginosa strain (C1712M) from a cystic fibrosis patient. In contrast, the same nonmucoid strain (PA-96), after exposure to PO2 of -40 mm Hg, showed little IF staining for alginate. Following enzyme treatment with alginase, PA-96 cells previously exposed to the higher PO2 and exhibiting enhanced alginate production, as determined by IF staining, now showed no IF staining. Moreover, treatment of the oxygen-up-regulated PA-96 cells with alginase released amounts of unsaturated alginate breakdown products (uronic acids) quantitatively similar to those released by typically mucoid strains treated with the same enzyme. These data indicated that the P. aeruginosa exopolysaccharide in our studies was, indeed, mucoid alginate and that variations in oxygen tensions represent one of the trigger mechanisms for the up-regulation of mucoid exopolysaccharide production.

There is a notable valve site-specific difference in the clinical outcomes of bacterial endocarditis cases, with patients experiencing left-side infection (aortic and mitral valves) having a significantly worse prognosis than those with right-side infection (1, 22). This disparity in endocarditis is most prominent with Staphylococcus aureus and Pseudomonas aeruginosa (1, 22, 23). The mechanism(s) underlying the differential outcome depending on valve site in endocarditis is undoubtedly multifactorial. We recently confirmed that exposing nonmucoid P. aeruginosa cells in broth in vitro to oxygen tensions reflective of the left-side cardiac circuit in vivo (PO2, -80 mm Hg) resulted in a substantial enhancement of exopolysaccharide production (4); in contrast, exposure of the same cells in vitro to oxygen tensions reflective of the right-side cardiac circuit in vivo (PO2, -40 mm Hg) resulted in no exopolysaccharide enhancement. In addition, scanning electron microscopy revealed cell surface excrescences on P. aeruginosa that were compatible with mucoid exopolysaccharide (MEP) in aortic valve vegetations but not in right-side tricuspid valve vegetations. Also, the presence of this oxygen-up-regulated exopolysaccharide correlated with the mitigation of aminoglycoside-induced growth inhibition and killing in vitro and ex vivo (4). Despite these provocative observations, that study did not confirm the enhanced exopolysaccharide to be P. aeruginosa mucoid alginate, since nonspecific polysaccharide stains (periodic acid-Schiff and ruthenium red) were used. The current study was thus designed to elucidate by immunofluorescence (IF), enzymatic, and biochemical strategies whether the oxygen*

up-regulated exopolysaccharide in nonmucoid P. aeruginosa strains was in fact MEP. (This study was presented in part at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, Tex., 17 to 20 September 1989 [A. S. Bayer, F. Eftekhar, D. Speert, and C. C. Nast, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 977, 1989].) MATERIALS AND METHODS Bacterial strains. The P. aeruginosa strains used in this study included PA-96, a nonmucoid isolate demonstrating

oxygen-up-regulatable exopolysaccharide production (this strain has been used in a number of prior experimental endocarditis studies in our laboratory [4, 5]); 144MR (a mucoid strain isolated from a patient with cystic fibrosis) and its isogenic, nonmucoid revertant, 144NM (kindly provided by Neal Schiller, Riverside, Calif. [6]); and strain C1712M (a mucoid strain isolated from a child with cystic fibrosis [10]). All strains were stored at -70°C in brain heart infusion broth (BHIB; Difco Laboratories, Detroit, Mich.) with 8% dimethyl sulfoxide until subcultured into fresh BHIB for use in this investigation. Alginase preparation. The alginase used in this study was obtained from Bacillus circulans (ATCC 15518) by a previously detailed technique (10). Briefly, B. circulans was grown in alginate-yeast extract medium for 3 to 4 days, and the culture supernatant was concentrated by microfiltration. After ammonium sulfate precipitation and dialysis, the crude enzyme preparation was frozen at -20°C until thawed for use on the day of the study. The specific activity of the crude alginase enzyme preparation was delineated by measuring

Corresponding author. 1344

PSEUDOMONAL ALGINATE PRODUCTION

VOL. 58, 1990

the yield of unsaturated material after enzyme action on alginate substrates by the periodate-thiobarbituric acid procedure of Weissbach and Hurwitz (28). Crude alginase was added to sodium alginate (2.5 mg/ml) in a volume of 500 [lI. Fifty-microliter samples were taken at 10-min intervals over 30 min and tested in the thiobarbituric acid assay. One unit of enzyme activity was defined as the amount of enzyme required to release an equivalent of 1 nmol of ,B-formylpyruvate (BFP) per min per ml; 0.01 ,umol of BFP produces an increase in the optical density at 549 nm of 0.29. The specific activity of the alginase enzyme, expressed as units of enzyme activity per milligram of protein (determined by the Lowry technique [16]), was 7.7. Alginase treatment of P. aeruginosa cells. The P. aeruginosa strains to be tested were grown in BHIB to a concentration of -109 CFU/ml, as determined spectrophotometrically. Bacterial cells were pelleted by centrifugation, washed twice in phosphate-buffered saline (PBS), and suspended to -109 CFU/ml, as determined spectrophotometrically, in PBS. The bacterial suspensions were incubated with alginase (125 U/ml; specific activity, 7.7), heat-inactivated (60 min for 2 h) alginase, or PBS (control) for 90 min at 37°C in a total volume of 500 ,ul. At the end of the 90-min incubation, the samples from the various reaction mixtures were centrifuged and the supernatants were tested in the thiobarbituric acid assay. The quantitative effect of alginase on the different P. aeruginosa strains was determined by measuring the nanomoles of BFP released into the supematant over the 90-min reaction period. In parallel studies, samples from the various reaction mixtures were removed, the alginase-treated or untreated P. aeruginosa cells were pelleted by centrifugation, washed, and resuspended in PBS, and the samples were smeared on glass slides for IF microscopy as described below. Indirect IF of P. aeruginosa strains with and without alginase pretreatment. Air-dried, heat-fixed smears of P. aeruginosa cells either pretreated or not with alginase were made on glass slides from the various reaction mixtures described above. A protein A-affinity-purified mouse monoclonal antibody against MEP was used in this study (kindly provided as a gift by Gerald Pier, Harvard University, Boston, Mass.); the specificity of this antibody against MEP has been confirmed (20). Preliminary IF screening with a mucoid P. aeruginosa control strain in our laboratory demonstrated that a 1:2 dilution of the monoclonal antibody in PBS (pH 7.4) with 1% fetal calf serum gave the best IF results. The bacterial smears were incubated with the monoclonal antibody or PBS at room temperature for 30 min and washed three times with PBS-fetal calf serum. Fluoresceintagged goat anti-mouse immunoglobulin G (Organon Teknika, Malvern, Pa.) was added to each smear at a dilution of 1:20, and the slides were incubated at room temperature for another 30 min. The slides were washed four times with PBS-fetal calf serum, air dried, and examined with a Zeiss epifluorescence microscope. The intensity of the IF was graded from 0 (no or minimum staining) to 4+ (maximum staining) by one of us (C.C.N.) without knowledge of either the pseudomonal strain identity or the use of alginase pretreatment. Growth of nonmucoid P. aeruginosa strains at different oxygen tensions. To confirm the presumed identity of the oxygen-up-regulated exopolysaccharide of the nonmucoid strain PA-96 as MEP, we grew this strain in the presence of pO2s of -40 or 80 mm Hg; these oxygen tensions were selected to approximate those observed in the right and left ventricles of the heart, respectively. Briefly, PA-96 was

1345

TABLE 1. IF of P. aeruginosa strains determined with an anti-alginate monoclonal antibody with and without alginase pretreatment Condition

Qualitative IF

PA-96a PA-96 PA-96

P02, 80 mm Hg P02, 40 mm Hg P02, 80 mm Hg; alginase added

4+ 0

C1712Mb C1712M

Alginase added

Strain

144MRb 144NMa 144MR a

b

Alginase added

0 4+ 0 4+ 0 0

Phenotypically nonmucoid. Phenotypically mucoid.

grown overnight without agitation in BHIB at 37°C. The growth sample was split, and P. aeruginosa cells were exposed for 6 h at 37°C to a P02 of either -40 or -80 mm Hg. A P02 of -40 mm Hg was achieved as previously described (4) by bubbling a gas mixture of 6% 02-94% N2 (Liquid Carbonic, Los Angeles, Calif.) into PA-96-containing BHIB for 5 min, after which the culture tube was sealed with paraffin and placed in a candle jar. Ambient air within the jar was flushed out with the 6% oxygen gas mixture. To achieve sustained P02S of .80 mm Hg, we followed the same protocol as that described above for the 6% oxygen gas mixture but substituted bubbled ambient air (PO2, -140 mm Hg) for the 6% oxygen gas mixture. We have confirmed by direct oximetric studies that these different in vitro exposures yield the desired oxygen tensions during the bubbling process within broth containing growing P. aeruginosa cells (4). PA-96 cells exposed to the two different oxygen tensions were grown for 6 h at 37°C prior to sampling for IF, enzymatic, and biochemical studies. We have confirmed that PA-96 cells grown for 24 h at 37°C following exposure to these two oxygen tensions have virtually identical growth curves (4). RESULTS IF studies with and without alginase pretreatment. Table 1 summarizes the results of the IF study with the specific anti-MEP murine monoclonal antibody. Untreated, control mucoid P. aeruginosa C1712M and 144MR demonstrated maximal staining by IF (4+), while an untreated, control nonmucoid strain (144NM) demonstrated no IF staining. PA-96 cells (intrinsically nonmucoid by phenotype) showed maximal IF staining following 6 h of exposure in vitro to oxygen tensions of -80 mm Hg (Fig. 1); in contrast, the same strain showed little IF staining following in vitro exposure to lower oxygen tensions (-40 mm Hg). All strains showed negative IF when PBS was substituted for the murine monoclonal antibody in this study. After alginase pretreatment of mucoid strains C1712M and 144MR, IF staining with the anti-MEP murine monoclonal antibody was completely abrogated; similarly, alginase pretreatment of PA-96 cells following 6 h of exposure to oxygen tensions of -80 mm Hg eliminated IF staining with the anti-MEP murine monoclonal antibody. Quantitative release of alginate substrate by alginase. Figure 2 graphically summarizes the alginase-mediated alginate hydrolysis determined by measurement of the quantitative release of BFP. The two control mucoid strains (C1712M and 144MR) released substantial amounts of BFP following

1346

BAYER ET AL.

INFECT. IMMUN.

FIG. 1. IF staining of bacterial colonies of various P. aeruginosa strains with a murine anti-alginate monoclonal antibody. (a) Strain 144NM (nonmucoid control). (b) Strain 144MR (mucoid strain; positive control). (c) Strain PA-96 exposed to oxygen tensions of approximately 80 mm Hg. (d) Strain PA-96 exposed to oxygen tensions of approximately 80 mm Hg and then pretreated with alginase prior to IF staining. Note the elimination of positive staining by the enzyme pretreatment. (e) Strain PA-96 exposed to oxygen tensions of approximately 40 mm Hg. Note that there was no IF staining. Bars, 10 p.m.

alginase exposure (means, 10.3 and 6.357 nmol/90 min, respectively), while the control nonmucoid strain (144NM) released very small amounts of alginate substrate products (mean, 0.56 nmol/90 min of alginase exposure). Following alginase exposure, PA-96 cells preexposed to oxygen ten-

sions of -80 mm Hg released amounts of BFP similar to those released by the control mucoid strains (mean, 7.21 nmol/90 min), while PA-96 cells preexposed to lower oxygen tensions (-40 mm Hg) released little BFP (mean, 0.56 nmol/90 min). Heat inactivation of the alginase prior to use eliminated BFP release from mucoid and oxygen-treated, nonmucoid P. aeruginosa cells. To demonstrate the enzymatic nature of the reaction of MEP (alginate) with alginase, we examined the release of BFP over time from both commercially available sodium alginate (Sigma Chemical Co., St. Louis, Mo.) and MEP extracted from one of the mucoid strains in this study (144MR), as well as from whole 144MR cells. MEP extraction was performed by a 95% cold ethanol precipitation technique as previously described (27). Alginase-mediated BFP release was rapid and linear initially from both whole

PSEUDOMONAL ALGINATE PRODUCTION

VOL. 58, 1990

1347

(I)

CI,

15

0

03 0)

10

Q)

E

~0E

5

cp 'I

0~

0

IL

C1712 M

m

144R

144NM

PA-96

PA-96

(Po280) (PO240)

(7)

(6)

(6)

(9)

(4)

NUMBER OF EXP'MTs

FIG. 2. Hydrolysis of alginate substrates contained in the whole cells of various P. aeruginosa strains by alginase. The thiobarbituric acid method was used to measure BFP released. Error bars represent means ± standard errors of the means. EXP'MTs, Experiments.

cells of strain 144MR (0 to 30 min) and MEP extracted from strain 144MR (0 to 15 min) and reached a plateau thereafter (Fig. 3); for BFP release from purified alginate, the enzymatic reaction continued linearly over the entire 60-min sampling period (data not shown).

DISCUSSION

The MEP of P. aeruginosa is a loosely adherent extracellular glycocaalyx of polyuronic acid complexes composed of 1,4-linked D)-mannuronic and L-guluronic acids, which are

LL

D Cl) 0 12a_ 11x

0

LLU 10LUI J) 9-

*

14k4MR - EXTRACTED MEP

14t4MR - WHOLE CELLS

z CD

8-

7

:2 blJ

654-

:2

3-

LU

U)

2-

LL

I1-

',I

Lm 0 LL.

U1 -(IU

I ,~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5 10 15 20 25 30 35 40 45 50 55 60 65

* 70

75

I

I

---

80 85 90

TIME (MIN)

FIG. 3. Enzymatic hydrolysis of extracted P. aeruginosa MEP (mean of two experiments) and whole-cell MEP (mean of four experiments) by alginase. Error bars represent means + standard errors of the means. nM, Nanomoles.

1348

BAYER ET AL.

variably 0 acetylated in different strains (11, 25). The MEP of P. aeruginosa is produced in large quantities in phenotypically mucoid strains such as those that colonize the airways in children and adults with chronic cystic fibrosis (18). P. aeruginosa MEP has been shown to have several important biological functions, including mediation of attachment of mucoid strains to tracheal epithelium (21); resistance of mucoid strains to nonopsonic phagocytosis by monocytederived macrophages and neutrophils (8); mitigation of antibiotic (aminoglycoside) - diffusion in vitro, presumably through a barrier mechanism (17, 26); and resistance to oxidative, intracellular killing via the myeloperoxidase system, presumably by the ability of MEP to participate in hypochlorite scavenging (15). Anastassiou et al. (2) and others (19) have confirmed that MEP production is a property shared by both phenotypically mucoid and phenotypically nonmucoid P. aeruginosa strains and that both phenotypic variants contain the genetic information necessary for MEP production. A number of conditional triggers needed for phenotypically nonmucoid strains of P. aeruginosa to show enhanced MEP production, such as nutrient media enriched with magnesium and/or gluconate supplementation (7), aeration of growing cultures (14), and restriction of nutrients such as iron (3, 27), have been identified. Elegant studies by Goldberg and Ohman (13) and Flynn and Ohman (12) have shown that the regulatory and biosynthetic pathways for alginate production are complex, involving a number of widely dispersed genes, of which the algB gene is important in the efficient regulation of MEP production, while the algS gene is the putative genetic switch mechanism for MEP production. We have recently confirmed that different oxygen tensions may serve as another trigger mechanism for the up-regulation of MEP production by nonmucoid P. aeruginosa strains (4). We observed a substantial enhancement of exopolysaccharide production in an endocarditis-inducing, nonmucoid P. aeruginosa strain exposed in vitro to oxygen tensions reflective of the left-side (but not right-side) cardiac circuit in vivo. We additionally noted an enhancement of in vivo exopolysaccharide production in nonmucoid PA-96 cells infecting left-side (but not right-side) experimental cardiac vegetations. Moreover, these oxygen-up-regulated P. aeruginosa cells were substantially more resistant in vitro and ex vivo to the growth-inhibitory and bactericidal effects of aminoglycosides, as characterized by postantibiotic effect and time kill curve methods, respectively. We theorized that this up-regulation of exopolysaccharide production at higher oxygen tensions may underlie the relatively poor therapeutic outcomes seen in left-side P. aeruginosa endocarditis, similar to the inferior treatment outcome in viridans streptococcal endocarditis caused by exopolysaccharide (dextran)producing strains (9). The current study, investigating oxygen-dependent upregulation of P. aeruginosa exopolysaccharides, produced several interesting observations. By IF staining with a murine monoclonal antibody, we confirmed that the oxygenenhanced exopolysaccharide seen in nonmucoid P. aeruginosa cells was, indeed, MEP. The qualitative IF staining characteristics for this oxygen-exposed nonmucoid P. aeruginosa strain were very similar to those observed for two phenotypically mucoid variants. Moreover, the identification of the oxygen-up-regulated exopolysaccharide as MEP was further supported by the ability of alginase enzymatic treatment to (i) qualitatively remove MEP and render the strain IF negative and (ii) release amounts of alginate substrate products quantitatively very similar to those re-

INFECT. IMMUN.

leased from typically mucoid strains. Heat inactivation of the enzyme negated these latter effects. Sengha et al. (24) have recently shown that the production of alginate by P. mendocina in vitro under nitrogen-limited conditions is optimal at a P02 of -40 mm Hg; this P02 approximates that remaining in log-phase cultures of our P. aeruginosa strain (PA-96) initially exposed to a P02 of -80 mm Hg at the time of maximum exopolysaccharide production by this organism (4). This fall in oxygen tension from an initial P02 of -80 is felt to be due to bacterial respiration (4). The aortic valve vegetation in endocarditis is a protected focus for the unbridled proliferation of bacteria, related in part to limited phagocytic cellular host defenses (especially granulocytic ones) operative at this valvular site (5). Our prior (4) and current studies support the concept that the upregulation of bacterial exopolysaccharide may also play a role in preventing effective intravegetation bacterial clearances and suggest a potential role for enzymatic (e.g., alginase) strategies in this regard. Alginase enzymes alter MEP to render the mucoid P. aeruginosa cell surface susceptible to nonopsonic phagocytosis and killing by macrophages and neutrophils (10). Also, we have shown that such enzymatic pretreatment improves the ability of aminoglycoside antibiotics to kill mucoid P. aeruginosa strains in vitro (S. Park, J. Tu, D. P. Speert, A. S. Bayer, Clin. Res., in press). Lastly, there is an in vivo precedent for the use of enzymatic strategies to enhance the clearance of exopolysaccharide-producing bacterial strains from experimentally infected animals. Dall et al. (9) recently showed that a specific dextranase could remove the surface glycocalyx from dextran-positive viridans streptococci and facilitate penicillin-induced sterilization of aortic valve vegetations caused by such strains. We are currently pursuing such in vivo enzymatic strategies in relation to MEP-producing P. aeruginosa strains. ACKNOWLEDGMENTS This research was supported in part by a research grant (SJ5993-03) from the Saint John's Heart Institute, Santa Monica, Calif. (to A.S.B.), and research grants from the Medical Research Council of Canada and the Canadian Cystic Fibrosis Association (to D.P.S.). LITERATURE CITED 1. Abrams, B., A. Sklaver, T. Hoffman, and R. Greenman. 1979. Single or combination therapy of staphylococcal endocarditis in intravenous drug abusers. Ann. Intern. Med. 90:789-791. 2. Anastassiou, E. D., A. S. Mintzas, C. Kounavis, and G. Dimitracopoulos. 1987. Alginate production by clinical nonmucoid Pseudomonas aeruginosa strains. J. Clin. Microbiol. 25:656659. 3. Anwar, H., T. Van Biesen, M. Dasgupta, K. Lam, and J. W. Costerton. 1989. Interaction of biofilm bacteria with antibiotics in a novel in vitro system. Antimicrob. Agents Chemother.

33:1824-1826.

4. Bayer, A. S., T. O'Brien, D. C. Norman, and C. C. Nast. 1989. Oxygen-dependent differences in exopolysaccharide production and aminoglycoside inhibitory-bactericidal interactions with Pseudomonas aeruginosa-implications for endocarditis. J. Antimicrob. Chemother. 23:21-35. 5. Bayer, A. S., J. Yih, C. Y. Chiu, and C. C. Nast. 1989.

Pathogenic effects of monocytopenia, granulocytopenia and dexamethasone on the course of experimental Pseudomonas aeruginosa endocarditis in rabbits. Chemotherapy 35:278-288. 6. Borowski, R. S., and N. L. Schiller. 1983. Examination of the bactericidal and opsonic activity of normal human serum for a mucoid and nonmucoid strain of Pseudomonas aeruginosa. Curr. Microbiol. 9:25-30. 7. Buckmire, F. L. A. 1984. Influence of nutrient media on the

PSEUDOMONAL ALGINATE PRODUCTION

VOL. 58, 1990

8.

9.

10.

11.

12.

13.

14. 15.

16. 17.

18.

characteristics of the exopolysaccharide produced by three mucoid Pseudomonas aeruginosa strains. Microbios 41:49-63. Cabral, D. A., B. A. Loh, and D. P. Speert. 1987. Mucoid Pseudomonas aeruginosa resists nonopsonic phagocytosis by human neutrophils and macrophages. Pediatr. Res. 22:429-431. Dali, L., W. G. Barnes, J. W. Lane, and J. Mills. 1987. The enzymatic modification of glycocalyx in the treatment of experimental endocarditis due to viridans streptococci. J. Infect. Dis. 156:736-740. Eftekhar, F., and D. P. Speert. 1988. Alginase treatment of mucoid Pseudomonas aeruginosa enhances phagocytosis by human monocyte-derived macrophages. Infect. Immun. 56: 2788-2793. Evans, L. R., and A. Linker. 1973. Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J. Bacteriol. 116:915-924. Flynn, J. L., and D. E. Ohman. 1988. Cloning of genes from mucoid Pseudomonas aeruginosa which control spontaneous conversion to the alginate production phenotype. J. Bacteriol. 170:1452-1460. Goldberg, J. B., and D. E. Ohman. 1987. Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate. J. Bacteriol. 169:1593-1602. Krieg, D. P., J. A. Bass, and S. J. Mattingly. 1986. Aeration selects for mucoid phenotype of Pseudomonas aeruginosa. J. Clin. Microbiol. 24:986-990. Learn, D. B., E. P. Brestel, and S. J. Mattingly. 1986. Hypochlorite scavenging by Pseudomonas aeruginosa alginate. Infect. Immun. 55:1813-1818. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Nickel, J. C., I. Ruseska, J. B. Wright, and J. W. Costerton. 1985. Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob. Agents Chemother. 27:619-624. Pennington, J. E., S. M. Wolff, and P. Puziss. 1979. Summary of

19.

20.

21.

22. 23.

24. 25.

1349

a workshop on infections in patients with cystic fibrosis. J. Infect. Dis. 140:252-256. Pier, G. B., D. Desjardins, T. Aguilar, M. Barnard, and D. P. Speert. 1986. Polysaccharide antigens expressed by nonmucoid isolates of Pseudomonas aeruginosa from cystic fibrosis patients. J. Clin. Microbiol. 24:189-196. Pier, G. B., J. M. Saunders, P. Ames, M. S. Edwards, H. Auerbach, J. Goldfarb, D. P. Speert, and S. Hurwitch. 1987. Opsonophagocytic killing antibody to Pseudomonas aeruginosa mucoid exopolysaccharide in older noncolonized patients with cystic fibrosis. N. Engl. J. Med. 317:793-798. Ramphal, R., and G. B. Pier. 1985. Role of Pseudomonas aeruginosa mucoid exopolysaccharide in adherence to tracheal cells. Infect. Immun. 47:1-4. Reyes, M. P., and A. M. Lerner. 1983. Current problems in the treatment of infective endocarditis due to Pseudomonas aeruginosa. Rev. Infect. Dis. 5:314-321. Sande, M. A., and 0. M. Korzeniowski. 1981. The antimicrobial therapy of staphylococcal endocarditis, p. 113-122. In A. L. Bisno (ed.), Treatment of infective endocarditis. Grune & Stratton, New York. Sengha, S. S., A. J. Anderson, A. J. Hacking, and E. A. Dawes. 1989. The production of alginate by Pseudomonas mendocina in batch and continuous culture. J. Gen. Microbiol. 135:795-804. Sherrock-Cox, V., N. J. Russel, and P. Gacesca. 1984. The

purification and chemical characterization of the alginate present in the extracellular material produced by mucoid strains of Pseudomonas aeruginosa. Carbohydr. Res. 135:147-154. 26. Slack, M. P., and W. W. Nichols. 1981. The penetration of antibiotics through sodium alginate and through the exopolysaccharide of a mucoid strain of Pseudomonas aeruginosa. Lancet ii:502-503. 27. Speert, D. P., S. W. Farmer, M. E. Campbell, J. M. Musser, R. K. Selander, and S. Kuo. 1990. Conversion of Pseudomonas aeruginosa to the phenotype characteristic of strains from patients with cystic fibrosis. J. Clin. Microbiol. 28:188-194. 28. Weissbach, A., and J. Hurwitz. 1959. The formation of 2keto-3-deoxyheptonic acid in extracts of Escherichia coli B. I. Identification. J. Biol. Chem. 234:705-709.