Scanning Electron Microscopic Study of Uropathogen Adherence to a Plastic Surface

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1983, p Vol. 45, No /83/ $02.00/0 Copyright 1983, American Society for Microbiology Scanning Electron Microscopic Study of Uropathogen Adherence to a Plastic Surface THOMAS J. MARRIE' 2* AND J. W. COSTERTON3 Departments of Medicine1 and Microbiology,2 Dalhousie University and the Victoria General Hospital, Halifax, Nova Scotia, Canada B3H 1 V8, and Department of Biology, University of Calgary, Calgary, Alber-ta, Canada T2N IN43 Received 7 June 1982/Accepted 23 December 1982 We used a polyethylene surface to study the adherence of various urinary pathogens to a representative inert surface. The bacteria were suspended in filtersterilized urine during this adhesion study, and differential adhesion was clearly demonstrated. Pseudomonas aeruginosa adhered most avidly and formed large microcolonies that were surrounded by an extensive amorphous matrix. Staphylococcus saprophyticus also formed microcolonies on the surface of the plastic droppers. In general, piliated strains of Escherichia coli adhered less avidly than the other organisms, but more avidly than nonpiliated strains; however, one piliated strain of E. coli adhered very poorly and behaved like a nonpiliated strain. The adherence of bacteria to the surfaces of mammalian cells has been studied extensively in recent years. A consensus is developing that adhesion is important as a virulence factor in the establishment of infection (8, 17, 19) and that attributes of both the host (8) and the microorganisms (4) are important in this process. Specific ligands have been described that mediate this adhesion, such as the mannose-sensitive pili of many strains of the Enterobacteriaceae (14), and some organisms have been found to possess multiple adherence mechanisms. An increasing proportion of infections are associated with the prosthetic devices (9) that are used to treat seriously ill patients, and these pathogens may be introduced with the device or may grow on the surface of the device itself. Infections involving prosthetic devices are very difficult and often impossible to cure with antibiotics alone (21), and surgical removal is frequently necessary (5). The study of bacterial attachment to some of the inert materials used in humans revealed differential adhesion-staphylococcus aureus adhered better to gut than to silk or nylon sutures (18). In a study of catheteracquired bacteriuria, we found extensive development of a biofilm containing bacteria on the surface of the reservoir bags of these systems (submitted for publication). To study the kinetics of such attachment, we used scanning electron microscopy (SEM) to examine the surfaces of plastic droppers exposed to urine containing various uropathogens. MATERIALS AND METHODS Organisms. Four strains of nonpiliated and six strains of piliated Escherichia coli isolated from the midstream urine specimens of patients with symptomatic bacteriuria were used. The presence or absence of pili was determined by the method of Svanborg-Eden and Hansson (20). One strain of Pseudomonas aeruginosa (ATCC 25619) was obtained from the American Type Culture Collection, Cockeysville, Md. The strain of Staphylococcus saprophyticus used was isolated from the midstream urine sample of a young female suffering from frequent and urgent urination and dysuria. All isolates from urine were subcultured twice before use in these experiments. Attachment of microorganisms to plastic droppers. Each strain was grown on blood agar plates (Trypticase soy agar, BBL Microbiology Systems, Cockeysville, Md.) containing 5% sheep blood and then inoculated into tryptose phosphate broth and incubated at 35 C for 6 h. The inoculum was adjusted to the density of one-half MacFarland standard. A 1-ml amount (5 x 107 cells ml- ') of each microorganism was added to 10 ml of filter (Millipore Corp., Bedford, Maine) in a 60- ml bottle-eye dropper assembly (Polyethylene Nalgene, with polypropylene screw closure and dropper assembly; Nalgene Corp., Rochester, N.Y.). The urine-filled dropper assemblies were held at room temperature, and once each day the dropper was briefly filled with contaminated urine and then emptied by squeezing the dropper bulb. Each day for 10 days, one dropper was removed from one of the four series containing piliated and three of the six series containing nonpiliated strains of E. coli and one from the series containing S. saprophyticus; the droppers were prepared for SEM as described below. The series of dropper-bottle assemblies containing P. aeruginosa was examined on days 2 and 10 only. Colony counts were performed on the urine in each bottle at the time the dropper was removed. A dropper-bottle containing only filter-sterilized urine without added microorganisms was included as a control. Three strains of piliated and three strains of nonpiliated E. coli were studied as described above, except that each was inoculated in duplicate to the bottle-eye 1018

2 VOL. 45, 1983 dropper assembly. One dropper of each set was fixed for SEM as described below, and the other was removed from the urine and rinsed in 5 ml of sterile brain heart infusion broth (Difco Laboratories, Detroit, Mich.) by aspirating the fluid up and down 50 times. Colony counts were performed on the rinse fluid. The inner surface of the dropper was then sampled with a cotton swab, which was used to inoculate a Trypticase soy agar plate containing 5% sheep blood, and the number of colonies was counted. SEM. Each dropper was placed in a fixative solution consisting of 5% glutaraldehyde in cacodylate buffer (0.067 M, ph 6.2) with 0.15% ruthenium red for 1 h at 20 C. This solution was changed three times at hourly intervals. The specimens were then metallized with osmium tetroxide and thiocarbohydrazide (11) and dehydrated in ethanol and Freon 113 before criticalpoint drying (3) and examined with a Hitachi S 450 (Hitachi, Rexdale, Ontario, Canada) scanning electron microscope. RESULTS The urine colony counts for all strains were o0.5 x 108 CFU/ml on day 1. The colony counts for four strains of E. coli had declined to 105 to ADHERENCE OF UROPATHOGENS CFU/ml by day 10; those for the other six strains remained at 108 CFU/ml. The urine colony counts for P. aeruginosa and S. saprophyticus remained at 108 CFU/ml throughout the 10 days of this study. The control unit remained sterile throughout the study, and the inner surface of this dropper, as seen at day 7, is shown in Fig. 1. Note the absence of bacteria on this relatively rough surface. All four of the nonpiliated strains of E. coli adhered very poorly to the surface of the dropper (Fig. 2). In contrast, five of the six piliated strains adhered well (Fig. 3). Furthermore, these five strains formed microcolonies on the dropper surface and seemed at times to be enclosed in an amorphous matrix (Fig. 3). Floccular material was also evident on the surface of these bacteria (Fig. 3), and these extracellular structures probably represent various condensed forms of the hydrated exopolysaccharides that surround the cells in life and are radically condensed during the dehydration steps of preparation for SEM FIG. 1. Scanning electron micrograph of the inner surface of a plastic dropper immersed in sterile filtered urine for 7 days. Note the absence of bacteria and the relatively smooth surface at low magnification (inset; bar, 50,um) and the uneven surface at higher magnification (bar, 5,um).

3 1020 MARRIE AND COSTERTON APPL. ENVIRON. MICROBIOL. FIG. 2. Scanning electron micrographs of the inner surface of a plastic dropper immersed in urine contaminated with a nonpiliated strain of E. coli for 7 days. Note the sparse colonization of this surface by bacterial cells at both low (inset) and high magnification. Bars, 5,um. (4). Condensed fibrous strands were seen to interconnect bacteria and anchor them to the plastic surface (Fig. 3). None of the nonpiliated strains formed a biofilm on the inner surface of the droppers. Piliated E. coli strain (Table 1) behaved like a nonpiliated strain in that it failed to form a biofilm of microorganisms on the inner surface of the dropper and adhered very poorly, as seen by SEM. The results of the quantitative cultures of the three piliated and three nonpiliated E. coli strains (Table 1) showed no differences in adhesion on day 1. Two of the three piliated strains and one of the two nonpiliated strains adhered very well by day 10. All six strains showed a considerable increase in adherence by day 10 as compared with day 1. P. aeruginosa had colonized the surfaces of the droppers very heavily on day 10 and had produced a continuous adherent biofilm composed of large microcolonies in an extensive amorphous matrix (Fig. 4A). Bacterial cells were seen to occupy the biofilm, so that several layers of cells of this organism were found on surfaces within a matrix that had condensed radically during dehydration in preparation for SEM. Cells of S. saprophyticus also adhered well to the surface of the plastic droppers to form small microcolonies composed of 6 to 10 bacterial cells (Fig. 4B). DISCUSSION The pronounced tendency of bacteria to grow in close association with surfaces in aquatic environments (22) may be explained in part by

4 VOL. 45, 1983 ADHERENCE OF UROPATHOGENS 1021 FIG. 3. Scanning electron micrographs of a plastic dropper immersed in urine contaminated with a piliated strain of E. coli for 7 days. Note that this plastic surface is more heavily colonized than that shown in Fig. 2; also note the presence of floccular material on the surface of the bacterial cells. Fine strands of exopolysaccharide material, which had condensed during specimen dehydration, were seen to interconnect bacterial cells and apparently to mediate attachment to these inert surfaces. Some microcolonies of adherent cells were clearly enclosed in a coherent, continuous, amorphous matrix of condensed exopolysaccharide, as shown here. Bar, 5 p.m (inset bar, 10 Rm). the observation of Nickels et al. (13) that aquatic bacteria produce biopolymers that coat submerged surfaces. This film attracts microbes that bind reversibly then irreversibly (12) to the surface and develop to form microcolonies within a continuous extracellular acid polysaccharide matrix (4). These extensive biofilms cause fouling of cooling systems (6), and their accumu-

5 1022 MARRIE AND COSTERTON APPL. ENVIRON. MICROBIOL. TABLE 1. Results of quantitative cultures of urine, broth used to rinse droppers, and cultures of swabs from the inner surface of droppers immersed in urine contaminated with various strains of E. coli Colony counts on indicated day for indicated source E. coli strain Day 1 Day 10 Urine Dropper rinsea Swab from inner surface Urine Dropper rinsea Swab from inner surface (CFU/ml) (CFU/ml) of dropper (CFU) (CFU/ml) (CFU/ml) of dropper (CFU) Piliated x >1, i08 6 x >1, x Nonpiliated X X a The dropper was recovered from the urine and rinsed in S ml of brain heart infusion broth by aspirating the fluid up and down 50 times. lation has even frustrated large-scale schemes, such as the ocean thermal energy conversion projects that depend on heat exchange across metal surfaces (1). Where possible, industry has used biocides, such as hypochlorite, that remove biofilms by oxidizing their polymeric matrices (2), and a new class of isothiazalone biocides has now been developed that penetrates the polymeric matrix of the biofilm to kill its component bacteria (16). Many of these same considerations of bacterial adhesion and colonization apply to the urinary catheters used in the treatment of patients, which can have serious sequelae in terms of infection (9). In spite of the obvious importance of the safe use of these devices, very little research has been done to find optimal materials and finishes or to develop safe biocides that would effectively prevent or minimize bacterial colonization. The urinary system incorporates one factor not found in natural and industrial systems-the glycosaminoglycan nonfouling polymers produced by the urinary bladder that have been shown to inhibit bacterial adherence (15). This work examined the differential tendency of uropathogens to adhere to a polyethylene surface in the form of an eye dropper assembly that was provided with a sterile closure and enclosed in a bottle that was partially filled with urine containing various bacteria. Polyethylene was chosen for these experiments because some of the urine droppers used clinically in our hospital are made from this material (others are glass), as are urinary catheter reservoir bags. Of the uropathogens tested, P. aeruginosa adhered most avidly and formed the most extensive glycocalyx (4) around its adherent microcolonies (Fig. 4A). This mode of growth was not unexpected, because this organism grows almost exclusively in adherent biofilms in its native habitat (7) in cold-water streams. The formation of microcolonies indicates that this organism grew actively after adhesion of cells from the urine milieu. Cells of S. saprophyticus adhered well to the polyethylene but less avidly than the pseudomonads, and they also formed microcolonies (Fig. 4B). In general, piliated strains of uropathogenic E. coli adhered to the polyethylene surface and formed small microcolonies surrounded by small amounts of glycocalyx, whereas the nonpiliated strains adhered only poorly and produced very little extracellular material. Our data also show the value of combining electron microscopic observations with quantitative cultures (Table 1), in that the one strain of piliated E. coli that adhered poorly as shown by SEM also adhered poorly as shown by quantitative cultures. In addition, this strain behaved like a nonpiliated one in that it produced no extracellular material. Table 1 also shows that adherence to these surfaces increased with time, and from the observation of microcolonies on the surfaces by SEM it was evident that growth occurred on the surface. Thus it is clear that uropathogens display a highly varied capacity to adhere to a polyethylene surface and that the production of extracellular polymers appears to be as important in the colonization of inert surfaces as it is in nature (4). The development of materials and biocides that limit the bacterial colonization of urinary prostheses will require a better understanding of the adherent mode of growth of uropathogens on inert surfaces.

6 VOL. 45, 1983 ADHERENCE OF UROPATHOGENS 1023 L-J FIG. 4. (A) Scanning electron micrograph of the inner surface of a plastic dropper immersed for 10 days in urine contaminated with P. aeruginosa. Note the heavy colonization of the plastic surface by microorganisms that have formed large multilayered microcolonies enclosed in an extensive matrix that collapsed during dehydration to produce enveloping amorphous masses. Bar, 5,um. (B) Scanning electron micrograph of the inner surface of a plastic dropper immersed for 7 days in urine contaminated with S. saprophvticus. Note the microcolonies composed of 6 to 10 coccoid cells. Bar, 5,um. ACKNOWLEDGMENTS Council of Canada and the Medical Research Council of We thank Joyce Nelligan and Carol Kwan for technical Canada. assistance and Daureen Stover for typing the manuscript. LITERATURE CITED This research was supported in part by grants from the 1. Aftring, R. P., and B. F. Taylor Assessment of Dalhousie University Internal Medicine Research Foundation microbial fouling in an ocean thermal energy conversion and from the National Science and Engineering Research experiment. Appl. Environ. Microbiol. 38:

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