The role of saliva in aggregation and adherence of Pseudomonas aeruginosa in patients with cystic fibrosis

S. K, Tumber-Saini', B, F, Habbick^ A, M. Oles\ J, P, Schaefer' and K, Komiyama^ Departments of 'Oral Biology. 'Community Health and Epidemiology. 'Royal University Hospital. University of Saskatchewan. Saskatoon. Canada

Tumber-Saini SK, Habbick BF, Oles AM, Schaclcr JP, Komiyama K: The role of saliva in aggregation and adherence of PsetuhnMnas aeruginosa in patients with cystic fibrosis. ,1 Oral Pathol Med 1992; 21: 299-304. The aggregation and adherence activity of P. aeruginosa, mediated by whole saliva from cystic fibrosis (CF) patients and non-CF subjects, was investigated. CF salivamediated aggregation of P. aerugino.su was stronger than the activity of non-CF saliva. Likewise, /' aeriigino.ta adherence to buccal epithelial cells (BEC) of CF" patients was stronger than to BEC of non-CF subjects. Adherence of non-mueoid P. aeruginosa to BEC of CF patients was increased by saliva, whereas the mucoid variant was not. CF patients colonized with /? aeruiiino.sa showed higher adherence of the non-mticoid variant than non-colonized CF patients. CF patients with high saliva-mediated adherence of non-mucoid P. aeruginosa also had high salivary aggregation activity. Increased CF saliva-mediated aggregation activity may be linked to the increased non-mueoid P. aeruginosa adherence to BEC of CF patients.

Cystic fibrosis (CF) is the most common autosomal recessive disease of Caucasians. It is characterized by chronic pulmonary infection, which is the major cause of morbidity and mortality. Fhe predominant pathogen responsible for pulmonary infeetion is PseudoDioiias aerfigino.sa (1 5). The reason for this predominanee is still not fully understood. It is well aeeepted that mierobial adherence to host tissues is a necessary prerequisite for their eolonization (6), Since oral colonization by P. aerugiito.sa in CF patients may play a significant role in the disease process (7-13), defining tbe factors involved in the process of colonization by this pathogen is important. Wooixs ef al. (14 16) demonstrated that decreased levels of cell surface fibronectin and increased levels of protease and pili were related to tbe adherence of P. aerugifiosa to buccal epithelial cells. However, the mechanisms by which /' aeiugifiosa adheres to CF" oral tissues have not beeti fully delineated. Human saliva contains several molecules which induce aggregation of oral bacteria. Salivary molecules implicated in the aggregation process inelude lysozyme (17, 18), .secretory IgA (19 21),

and high-molecular-weight glyeoproteins (22 24), We have recently found that saliva from CF' patients contained higher aggregating activity for P. aerugiffosa than did non-CF saliva and that the increased P. aerftgifw.sa aggregation seemed to be directly related to the sialic acid content in whole saliva, suggesting that tbis molecule acts as the salivary receptor for this pathogen (12). Furthermore, we have found that the seromucous products of the submandibular secretion may be responsible for tbe dilferences in /' aerughw.sa aggregation activity by saliva from CF patients and non-CF subjeets (13). Saliva-mediated aggregation of bacteria has been previously studied as a correlate of the bacterial adhesion promoting activity (25 29), The role of saliva-mediated aggregation in oral colonization of CF patients has yet to be determined. Therefore, the objective of this study was (a) to define tbe role of saliva in the adherence of mucoid and non-mucoid variants P. aefugifUKia to CF and non-CF buceal epithelial cells (BEC), and (b) to compare saliva-mediated /' (UTugino.sa aggregation and adherence promoting activities to define if a relationship exists between the two phenomena.

Key words: cystic fibrosis; P. aeruginosa. saliva Kunio Komiyama, Dept. of Oral Biology, College of Dentistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO Accepted for publication February 1, 1992.

Material and methods Subjects

A total of 16 CF patients (9 girls, 7 boys) attending the CF clinic at the Royal University Hospital (Saskatoon, Saskatchewan, Canada) participated in this study. The average age of the C F patients was 13,2 yr. The patients were in generally good health at the time of sampling and were not undergoing antibiotic therapy. Sixteen age- and sexmatched non-CF (NCF) subjects served as a control group. This investigation was approved by tbe University of Saskatehewan Advisory Committee on Ethics in Human Experimentation, and informed consent was obtained from tbe patients or from tbeir parents. Eacb individual in this study was examined for colonization by P aeruginosa. Sputum speeimens were eoUected in a sterile plastic container after tbe patient had coughed several times. Samples of oral tlora were taken from the buccal mucosa, tbe dorsum of the tongue, and saliva using separate sterile cotton swabs. Dental plaque satnples were obtained from the gingival margin using a sterile periodontal curette. Eacb specimen was plaeed into a tube eontaining 5 mL Tryptic soy broth (TSB;

300

TuMBER-SAiNf ef al.

Difco, Detroit, MI., U.S.A.) and kept on iee until processed in our laboratory. After dispersion of the samples from the swab or curette, a 0.1 mL aliquot of the specimen was plated onto Cetrimide agar (Difco) plates, in duplicate and incubated aerobically for 37 C. Standard criteria to identify P. aeruginosa were followed, as deseribed previously (11), Patients were identified as being colonized with P. aerugino.ia following isolation of the bacteria from any one of the sites sampled (buccal mucosa, tongue, dental plaque, saliva and/or sputum).

P. aeruginosa strains

Mucoid (TYTl) and non-mucoid (HSSl) strains of P aeruginosa of oral origin, which bad been previously isolated and eharacterized in our laboratory (11), were examined for their aggregation and adherence activity. These strains had been stored on serum-coated glass beads at - 7 0 C until needed (30), and were not further subcultured. There is evidenee to suggest tbat the physiology of laboratory strains transferred an unknown number of times in laboratories may not resemble that of freshly isolated strains (30-32). To avoid alteration of characteristics of the original isolates through in vitro transfer, these serum-coated glass beads were routinely used for subculturing (11-13). To prepare cultures for aggregation or adherence assays, beads were transferred into 5 mL TSB and incubated aerobically for 24 h at 37 C. The 5 mL sample was than transferred to 5 mL fresh TSB and reincubated. To prepare eultures for assays requiring radiolabelled cells, 5 mL of a 12-h culture of P. aeruginosa cells grown in TSB (approximately 10'' cells/ mL) was transferred to 15 mL fresh TSB containing 0.2 mL of uniformly labelled L-(U-"'C) Lysine-Monohydroehloride (50 fiCi/mL; Amersham International, Oakville, Ontario, Canada). The suspension was incubated at 37 C for 3 h in a shaking water bath (500 rpm). After the incubation period, tbe bacteria (labelled or non-labelled) were washed 3 times with O.I mol/L phosphate-buffered saline (PBS; pH 7.2) by centrifugation (10,000 x ^ for 10 min at 4 C) and finally resuspended in PBS at a concentration of 10'" cells/mL as determined spcetrophotometrically (absorbanee 1.5 at 700 nm). Absorbanee was correlated to bacterial cells/mL by using various MeFarland nephelometer barium sulfate standards (33). Different concentra-

tions of the mucoid and non-mueoid P. aeruginosa were then plated onto Cetrimide agar plate and absorbanee related to eells/mL graphically assessed.

Adherence assay

Tbe in vitro adherence of P. aeruginosa to BEC was examined using modified methods of Wooixs ef al. (14) and MCEACURAN & IRVIN (37). All assays

Collection of saliva

Paraffin-stimulated whole saliva was colleeted in ice-chilled vials. The saliva samples were heat-treated at 56 C for 30 min to inactivate any degradative enzymes, which may destroy the adherence or aggregating faetors (12, 13, 34, 35). The saliva was clarified by centrifugation at 10,000 x ^ for 10 min at 4 C. If not used immediately, saliva was stored at - 2 0 C. One freezing and tbawing of saliva was found to have little effect on its aggregation and/or adherence properties (12, 13). Collection of buccal epithelial celis

BEC were collected from CF atid nonCF subjects by vigorous swabbing of tbe buccal mucosa with a sterile, cottontipped swab. Tbe cells were dislodged from the swab by vortex agitation in 5 mL of PBS for 15 s. The cells were then washed three times by centrifugation (300 x ^ for 10 min at 4 C), followed by resuspension in 2 mL of PBS. The BEC concentration was determined by hemocytometer counts.

Aggregation assay

The saliva-mediated aggregation of P. aeruginosa was assessed by the turbidometric assay of ERtcsoN ef al. (36), Briefly, saliva (0.5 mL) was pipetted into small tubes and warmed for 5 min at 37 C. At the same time, but in separate tubes, the baeterial suspension (absorbanee 1.5 at 700 nm) was also warmed. In the tube containing the saliva, 0.5 mL of the bacterial suspension was added, as well as 0,5 mL PBS, The reaction was started by stirring the mixture on a vortex mixer for 10 s. The contents were transferred to a cuvette immediately and tbe absorbanee at time zero was measured at 700 nm, PBS was used as the reference blank. Cuvettes were maintained at 37 C for 90 min, at wbieb time a final reading was taken. Results of the aggregation assay were expressed as a percent aggregation after subtraction of a blank, obtained by incubating bacteria with PBS alone, to account for the effects of cells settling over time.

were performed in duplicate, A 0.2 mL aliquot of BEC suspension (5x10'' cells/mL) was added to eaeh 15 mL polystyrene tube with an equal volume of saliva, or PBS for the eontrols. The combinations of BEC-saliva were: (a) CF BEC and CF saliva; (b) NCF BEC and NCF saliva; (e) CF BEC and NCF saliva; and (d) NCF BEC and CF saliva. The mixture was incubated in a shaking water bath (500 rpm) for 90 min at 37 C. After the incubation period, 0,2 mL of '""C-labelled P. aeruginosa was added, and tbe mixture incubated for an additional 90 min. This mixture was then transferred through a filter (5 |.im pore size; MSI, Westborougb, MA., U.S.A.) and washed 3 times witb 5 mL PBS to remove cells of non-adherent bacteria. The filter was transferred to a seintillation vial, 10 mL of scintillation cocktail added (Phase combining system, Amersbam International), and counts per minute (CPM) assessed using a LKB Rackbeta liquid scintillation counter. For each assay, 0,2 mL sample of the '•"C-labelled P. aeruginosa was added to scintillation vials and speeific activity determined. The cells typically bad a specific activity of approximately l - 2 x 10 ''CPM per colony forming unit. The number of bacteria attaehed per BEC was ealeulated by dividing the buccal-/? aeruginosa CPM by tbe specific activity of tbe 0.2 mL sample of '''C-labelled P. aeruginosa. The final BEC assoeiated radioactivity was expressed as the number of bacteria per BEC that adhered during incubation. Controls included incubating the BEC with PBS (instead of saliva), adding '""C-bacteria, and determining CPM. This allowed for baeteria attacbing to BEC in the absence of saliva to be subtracted from the assay results, to detect the effect of salivary molecule in the adherence process. Also, P. aeruginosa and saliva were incubated together without BEC, as a control for tbe aggregating effect affecting the filtering process and the test results. These latter control values were negligible indicating that the effect of the salivary-bacteria interaction on tbe final result was minimal. The results of non-speeifie binding of P. aerugino.sa to the filter was also determined as a control, using P. aeruginosa alone in the

CF saliva and P. aeruginosa adherence

assay. To correct for non-speeifie binding (generally very low), the resulting CPM was subtracted from the test assay, as well as the above controls. Microscopic observation using nonradiolabelled P. aeruginosa was performed to ensure that the adherenee assay process actually measured attachment of P. aeruginosa cells to the BEC. Tbe BEC-saliva and eontrols were set up as in the adherenee assay described above. After the filtration step, the filter was vortexed in 5 mL of PBS to remove adherent BEC and any free bacteria for examination. This proeedure was performed to eonfirm that P. aerugifwsa BEC attachment was measured by the procedure and not P. aeruginosa elumping due to salivary aggregation. The cells were incubated with 200 nL of 0.33% neutral red in Hanks Balaneed Salt Solution (Flow Laboratories Inc., Mississauga, Ontario, Canada) for 10 min. The suspension was centrifuged at 300 x^ for 10 min at 4 C and resuspended in 200 HL of PBS. A 200 |iL aliquot of trypan blue was added and the mixture ineubated for an additional 10 min; this was followed by tbree washes in PBS. After addition of 200 nL of glutaraldebyde (25% aqueous solution; grade II; Sigma, St. Louis, MO., U.S.A.), a sample was taken and added to a grid petri dish for eounting cells. The results of examining BEC, supernatants and the filters at different stages of the assay, indicated that the methodology followed in the adherence assay would allow for '''C-labelled P. aeruginosa attaehment, in the presence and absence of salivary molecules, to be measured.

tween salivary aggregation and adherence processes exists. Spearman Rank Correlation analysis was applied. Results

The role of saliva in the adherenee of P. aeruginosa to BEC from CF and nonCF subjeets was surveyed. Table 1 shows the number of mueoid (TYTl) and non-mueoid (HSSl) P. aeruginosa that adhered to CF BEC in the presenee and absence of saliva. For tbe different samples, saliva and BEC from the same individual were tested. The non-mucoid variant adhered better to BEC from CF patients than the mucoid variant. The non-mueoid variant adhered in significantly higher numbers to BEC ineubated witb saliva compared to BEC incubated without saliva (P = 0,01). The numbers of mueoid P aerugifw.ia tbat attached to BEC incubated witb or without saliva was not significantly different. A comparison was made of the adherence characteristics of the pathogen to BEC from nine CF patients colonized with P aerugifwsa (Group I) and to BEC from seven CF patients not eolonized with P. aerugiftosa (Group II) (Table 1). In the presence and absenee of saliva, tbe non-mucoid vari-

ant (HSSl) adhered significantly better to BEC from eolonized CF patients compared with BEC from non-colonized CF patients (P = 0.05. =0.01 respeetively). The attaehment of the mucoid variant (TYTl) to BEC from eaeh CF group was not signifieantly different. BEC reeovered from CF patients colonized with P. aeruginosa may bave already had the pathogen attaehed to tbe eells. Tbe extent of the effeet of subsequent in vitro attaehment processes could not be assessed. However, tbe results would probably show deereased attaehment of radiolabelled eells if BEC sites were already occupied and tberefore, the present observations would remain valid. Possible relationships between salivamediated aggregation and adherence processes were examined by Spearman Rank Correlation analysis. All CF saliva samples aggregated both mucoid and non-mucoid P. aeruginosa. CFsaliva had stronger non-mucoid aggregation activity compared with mucoid aggregation activity. Spearman Rank Correlation analysis showed signifieantly increased adherence of the tion-mueoid variant to CF BEC, in the presence of saliva, corresponding to high percent salivary aggregation aetivity of the nonmueoid variant by CF saliva (/'0.\0 in eacb instance). Differenees between CF and non-CF salivary adberence and aggregation activities of mucoid and non-mucoid P. aeruginosa are summarized in Table 3. A three way factorial ANOVA was used to test tbe differences between the following groups: CF and non-CF, mucoid and non-mucoid, incubation with and without saliva. Comparisons between groups were made using multiple range tests. As sbown in Table 3, adberence of non-mucoid and mucoid P. aerugino.sa to BEC from CF subjects was significantly bigber than to BEC from non-CF subjects, both in tbe presence and absence of saliva (P = 0.0\). CF saliva's percent aggregation of mucoid

Table 3, Comparison of mean adherence (±(SD) and percent aggregating activity of P aeruginosa in CF and non-CF subjects P aerugino.sa HSSl (non-mucoid)

PA/BEC

PA/BEC** Sample from CF* (n=16) non-CF (n = 16) P-value =

No saliva

With .saliva

15,1 ±4,0 20,9 ±7,3

P aerugino.sa TYTl (mucoid)

"A, Aggregation

No saliva

With saliva

Aggregation

26,8 ±9,9

10.8±3,5

8,6±3.2

15.1 ±9.6

••A,

4.0±l.l

3,8 ±1,3

5,9±3,1

4.3 ±3,3

3.8 ±3,2

5,8 ±5.0

0,01

O.OI

0.01

0.01

0.01

0,01

• CF samples include CI" patients colonized and not colonized with P. aerugino.ia ** Data are given as mean ± S D number of bacteria attached per epithelial cell SD = Standard deviation; PA = P aerugino.sa: BEC = Buccal epithelial cells

and non-mucoid P. aerugino.ia was also significantly bigber than by non-CF saliva {P = 0.0\).

BEC from CF colonized (Group I), non-eolonized (Group II) and NCF subjects were further assessed to determine the effect of saliva from four different sources (CF or NCF) on P. aerugino.ia attachment in tbe oral cavity. Table 4 sbows tbe sources of tbe BECsaliva that were examined. Adherence of non-mucoid P. aerugino.ia to Group 1 BEC, that were ineubated witb CF saliva, was signifieantly greater tban to Group 1 CF BEC ineubated with nonCF saliva (P = 0.01). Increased attachment of tbe non-mueoid variant was also found when NCF BEC were incubated witb Group I CF saliva eompared to NCF BEC incubated witb NCF saliva {P = 0.0\). No significant differences were found witb mueoid P aeruginosa attachment to tbe Group I CF BEC with CF or NCF .saliva. No .significant differences were found between the Group II BEC-saliva combinations. Incubating NCF BEC with the corresponding NCF saliva or with strongly aggregating CF saliva did not significantly increase tbe number of mucoid (TYTl) or non-mticoid (HSSl) P aerugino.ia tbat adbered to tbe BEC (Table 4). Discussion

Adherenee to host tissues is a necessary prerequisite for colonization by bacteria (6, 26), This study examined tbe role of saliva in this process, as well as the relationsbip, if any, between tbe P. aeruginosa aggregation and adberence processes in CF patients, Adberence of non-mucoid P. aeruginosa to CF" BEC was found to be significantly increased in tbe presence of saliva (Table I). There was less adberence, however, of tbe mueoid variant to BEC when CF saliva was added. Interestingly, it is tbe nonmucoid variant tbat bas been found to be tbe first type of P. iwruginosa to eolonize the oral eavities of CF" patients (2, 38, 39). We also found that adherence of tbe non-mucoid variant was particularly high in tbe CF patients already colonized witb P. aerugino.ia. These results Indicate tbat CF salivary secretions may be involved in P. aerugino.ia attachment to oral epithelial cells, particularly of the non-mucoid variant of P. aerugino.sa.

Our investigation also demonstrated tbat tbere was a positive eorrelation between CF saliva-augmented adherenee

CF saliva and P. aeruginosa adherenee 303 Table 4. Mean adherence ( + SD) of P. aerugino.sa to buccal epithelial cells when incubated ratory tract cells, mucoid variants may with CF saliva compared with non-CF saliva Sample from Group I* Group II

PA/CF BEC** P. aerugino.sa CF NCF Strain saliva saliva HSSl TYTl HSSl TYTl

P-value*

PA/NCF-BEC CF NCF saliva saliva

23.4 + 5.1 12,9+1 ,1 p = 0,01 16.6 + 9,7 9,7±4.4 15.2±4.6 I0.0±4 ,4 NS 16.9 + 5,0 14,0 + 5.6 6.4 + 2.7 NS 13.4 ±4,6 9,6 ±4 .5 NS 9.5 ±7,8

8.4+1,5 9.3 ±0,7 7.6 + 6.4

P- value* P = 0.05

NS NS NS

arise (2, 3, 37, 44, 45) and colonize the lower respiratory tract. Unfortunately advances in tbe fields of treatment and antibiotic tberapy in CF (46), have produced no means of eradicating P. aerugino.ia from the CF patient onee tbere is infection (8). VISHWANATH ef at. (39)

indicated tbat it may be the adhesionbost receptor interaction tbat may allow * Group I = CF patients colonized with P. aerugino.sa ('i = 4) for this pathogen to evade host defenses Group 1I = CF patients not colonized with P. aerugino.sa (H = 4) by (a) protecting against phagocytosis; ** Data are given as mean±SD number of bacteria attached per epithelial cell or (b) preventing physical clearance of * Three way factorial ANOVA with multiple range tests tbe mieroorganism once it is ancbored SD = Standard deviation; NS = Not significant; PA = f! aerugino.sa: BEC = Buccal epithelial to bost eells. Our results suggest tbat a eells part of the impairment of the CF host defense mechanism, is the failure of saliand aggregation activities of tbe non- NCF BEC (Table 3), Bacterial aggrega- vary aggregation to promote clearance mucoid variant of P. aeruginosa. but not tion by saliva was correspondingly of P. aeruginosa. Instead, saliva seems of the mucoid variant. This lends sup- found to be significantly higher in tbe to bave a role in adherenee of the nonport to the theory that in CF, the sali- CF group. This result is consistent witb mucoid variant to CF oral surfaces. We vary aggregation process may not aet our previous findings (12, 13). BEC of propose that, in CF, saliva-mediated agsolely as a mechanism for removal of CF patients colonized witb P. aentgino.ui gregation and adherence processes are this pathogen (12, 13). Instead, it indi- bad increased attachment when BEC important eomponents of P. aerugino.ia cates that there is something unique were pre-ineubated with the eorre- colonization of tbe oral cavity, and tbat about CF saliva and its aggregation sponding CF salivary secretions, com- such colonization may be a necessary characteristics that contributes to P aer- pared with CF BEC incubation witb prerequisite for subsequent lower respiuginosa eolonizing in these subjeets. A NCF saliva. However, BEC from CF ratory traet infeetion. Furtbermore, it is continuous source and large numbers of non-colonized patients or NCF subjects the altered salivary secretions which bacteria would need to be present in did not exbibit significantly altered P may be responsible for tbe differences saliva to avoid clearance mechanisms aerugino.sa attachment when BEC were observed in P. aeruginosa colonization (24, 25) and enhance tbe probability of incubated witb strongly aggregating CF in C F and non-CF individuals. host surface attachment (6, 26). It bas saliva eompared witb NCF saliva (Table been suggested that saliva-mediated ag- 4). Thus, while salivary constituents .•icknowledgemenfs - This investigation was gregation of bacteria may bave two dis- may contribute to tbe observed ditler- supported by a grant from the Canadian Cystinct functions. First, tbis activity may ences between tbe CF and NCF groups, tic Fibrosis Foundation to Drs, K. KOMIVApromote the clearance of bacteria by a variety of host and bacterial factors MA and B. F. HABBICK. We sincerely thank aggregating tbem into masses more easi- are also involved in inereased CF the CF patients for their time and patience, ly removed from tbe oral cavity (24-25). susceptibility to P. aerugifio.ia. Altered without which we would not have completed Second, this activity may promote the protease and fibronectin levels in CF this project, SHIRLEY PAroLA, B. Sc, R. N.; for her clinical assistance at the Cystic Fibroadberenee of baeteria to oral tissues and epithelial cells affect binding of P. aeru- sis Clinic; and ED ARMSTRONG, B. SC, M . SC. inhibited by salivary clearance mecha- gino.ia (14, 16). Pili are also important for his technical assistance. We also thank nisms (25-29). This latter suggestion surface structure components involved Dr. A. SI:NTHIL.SKIA'AN (Department of Comlends support to our bypotbesis tbat in P. aeruginosa attaebtnent to CF host munity Health and Epidemiology, College of saliva-mediated aggregation activity in surfaces (15). Tbe involvement of sali- Medicine. University of Saskatchewan) for CF patients may provide the numbers vary molecules as a facet of the bacteria- statistical analysis of data. of P. aeruginosa cells to increase the host surfaee interaetion has to date only availability of this pathogen in tbe oral been speculative. Our results suggest cavity and encourage its adherence. The that the role of saliva in increased P. 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