Journal of Microbiological Methods 10 (1989) 265-280 265 Elsevier

MIMET 00346

Kinetic measurement of bacterial adherence to eukaryotic cells

A . L . Smith 1, P.D. Jenny 1, L. Langley 2, P. Verdugo 2, M. Villalon 2, J. U n a d k a t 3 and D. Luchtel 4

IDivision of Infectious Disease, Children's Hospital and Medical Center; Department of Pediatrics, School of Medicine, 2Biological Structure; Center for Bioengineering, 3School of Pharmacy; 4School

of Public Health, University of Washington, Seattle, Washington, USA

(Received 9 June 1989; revision received 30 October 1989; accepted 3 November 1989)

Summary

We developed a kinetic assay using a monolayer of differentiated respiratory epithelium in culture to assess bacterial adherence. Mean residence time of bacteria in the tissue culture chamber was estimated from a model-independent (moment) analysis of the rate of bacterial washout from perfused Rose chambers. Results with this method compared favorably with visual assessment of adherence and the double radiolabel method with 14. influenzae. Adherence was assessed with low inoculae of 14. influenzae, P. cepacia and P.. aeruginosa avoiding cytotoxic effects seen when large inoculae are added to eukaryotic cells. This method will provide a means of assessing adherence of pathogenic respiratory bacteria to their cellular target at low inoculae.

Key words: Bacterial adherence; Respiratory epithelium

Introduction

Adherence of bacteria to surface epithelium is the first step (after acquisition) in the pathogenesis of certain infectious diseases. Colonization is facilitated by adherence and permits epithelial invasion or local toxin production: both can result in cellular injury. Bacteria adhere to epithelial surfaces by a variety of mechanisms. A low-affinity nonspecific surface structure can permit hydrophobic interaction with eukaryotic cell membranes [1]. A specific interaction between the bacterial surface and the mucosal surface is thought to be mediated by surface fimbriae [2]. Bacterial adhesins can be part of a distinct surface organelle (fimbriae) [3] and secreted into the media [4] or present as a continuous coat over the surface of bacteria [5]. Adhesins are thought

Correspondence to: A.L. Smith, Division of Infectious Disease, Children's Hospital and Medical Center, 4800 Sand Point Way NE/PO Box C5371, Seattle, WA 98105, USA.

0167-7012/89/$ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

266

to be responsible for organ tropism of certain bacteria and mediate high-affinity inter- action with a specific eukaryotic surface 'receptor' [2]. The species specificity of cer- tain mucosal pathogens (i.e., B. pertussis, N. gonorrhoeae and H. influenzae) is also thought to be due to their adhesins.

Bacterial adherence has been assessed in a variety of systems [6]. A commonly used technique harvests cells from a mucosal epithelium and mixes them with the test bac- teria. After 'washing' the eukaryotic cell suspension, the number of bacteria adherent to 100 epithelial cells is determined microscopically, by quantitative subcultures, or by determining the number of adherent radiolabelled bacteria. Disadvantages of visually quantitating adherence include using dead (buccal epithelial cells) or dying mammalian cells (easily desquamated bronchial cells, for example), and the assess- ment of only a small fraction of the eukaryotic cells in the assay: typically 20-100 of the 104 epithelial cells added are visualized and the adherent bacteria enumerated. To measure bacterial adherence with this method, large inocular (107-109 bacteria) are mixed with the epithelial cells. Large inoculae are necessary whether the adherent bacteria are enumerated visually [7, 8] or by contained radioactivity [9, 10]. Many microscopic fields have to be examined to enumerate a relevant number of bacteria and radiolabelling of bacteria with radioactive substrata rarely achieves a specific ac- tivity > 0.1 dpm. cell-l. Enumerating the bacteria by selective lysis of the eukaryotic cell and permitting the prokaryotic cells to form colonies also requires large inoculae. To determine a reliable estimate of bacterial density using standard 100-mm diameter plates, practical aspects of the method limit the plated bacterial density to 101 -102 cfu and volumes to 0.005- 0.5 ml. There are practical and theoretical disad- vantages of testing large inoculae. With bacteria colonizing the respiratory mucosa prior to invasion, such as capsulated H. influenzae, the inoculum acquired naturally is in the range of 104 -105 cfu. This estimate is derived from studies in infant primates in which bacterial density in nasopharyngeal washings averaged 6.2× 10 6 cfu.ml -l [11]. If you assume the wash procedure produced a 10-fold dilution, then the H. in- fluenzae b density is ~107 cfu.ml -~ of airway fluid; some fraction of this is then transmitted by coughing, sneezing or on fingers. Many bacteria, including Neiseria and Haemophilus, shed endotoxin-containing outer membrane blebs during late logarithmic growth. Endotoxin can have a cytotoxic effect on the target epithelia, resulting in adherence being assessed with viable and dying eukaryotic cells.

One indicator of bacterial adherence is the amount of shearing force required to detach the bacterium from its cellular target. Without a high-affinity ligand interacting with the bacterium, low-velocity fluid flow, such as tears, urine or saliva, should shear the bacterium from the mucosal surface. Ciliary activity effects movement of a mucous blanket over respiratory epithelium, an activity along with other flowing fluids, that bacterial adherence must overcome. We sought to devise an assay which would permit evaluation of adherence of bacteria pathogenic for the respiratory tract, to respiratory epithelia using low inoculae and in a flowing system. We assessed the average time of residence of a given bacterial strain in perfused respiratory epithelial monolayer cul- tures as an indicator of the strain's ability to resist shear forces.

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Materials and Methods

A. Tissue culture

1. Respiratory epithelia. Monolayers of respiratory epithelial cells were grown from mucosal sheets obtained from New Zealand white rabbits or pigtail monkeys Macaca nemestrina. Details of the tissue culture method have been published else- where [12]. Briefly, tracheal rings were repeatedly washed in sterile Hanks' medium and the mucosa excised by microsurgical procedures under a dissecting microscope. Small pieces of epithelium ~ 1 mm 2 were planted on glass coverslips placed on the bottom window of Rose chambers [13] and covered with an 8 #g-pore polycarbonate membrane. Rose chambers consist of two stainless steel sheets 1.8" thick measuring 3" × 1 ½ " with a 1" diameter circular hole in the center. Stainless steel screws are used to 'sandwich' a coverslip on each side of a Teflon gasket which is identical in shape to the steel sheets. Explants grow on a cellophane membrane which is between the coverslips.

2. Chang conjunctival cells. Monolayers of Chang conjunctival cells (ATCC CCL 202) were grown for 8 days in ATCC CRCM-30 media containing 10°70 fetal bovine sera (Hyclone, Provo, Utah), pH 7.4, at 37 °C. The culture media was supplemented with 2 mM glutamine (Gibco), 50 U .m1-1 of penicillin G, and 50/zg.ml -l of strep- tomycin. The cells were seeded on a removable plastic slide placed in le ighton tubes (Costar, Cambridge, Massachusetts). Prior to testing for adherence, the monolayers were washed twice with antibiotic-free and serum-free ATCC CRCM-30 medium.

B. Bacterial strains, media Type b H. influenzae Ela and its fimbriated derivative (designated R1369) have been

previously described [14]. H. influenzae undergoes phase variation from fimbriated to nonfimbriated at a frequency of 10 -4. Fimbriated H. influenzae agglutinate erythrocytes containing the Anton blood group antigen. Fimbriated derivatives can be selected from Fim- population by adsorption to Anton-positive erythrocytes. Fim ÷ H. influenzae also adhere to human buccal epithelial cells in greater numbers than F im- strains [9]. These H. influenzae strains were grown to the desired density in BHI broth supplemented with 10 #g.ml -l each of hemin hydrochloride, L- histidine and /3-nicotinamide adeninedinucleotide (Sigma Chemical, St. Louis, Missouri).

Pseudomonas cepacia (PC 109), isolated from the sputum of a CF patient, was kind- ly provided by Peter Gilligan, University of North Carolina, Chapel Hill, North Caroli- na. We derived a heavily fimbriated derivative (PC E213) from this isolate by repeated adsorption to human erythrocytes, in a manner similar to the procedure used for H. influenzae [14]. P. cepacia were grown to the desired density in M9 minimal media [15] supplemented with 0.2% casamino acids (BBL Microbiology Systems, Cockeysville, Maryland) and 2/~g. ml -~ glucose (0.2%); subcultures were performed on the same media solidified with 2.5o7o agar (BBL).

P. aeruginosa strain PAK (piliated) has been previously described [16]. P. aeruginosa

268

strain Psi is PAK with Tn5 inserted into the structural gene for the pilin subunit [17]: this strain was prepared and donated by Stephen Lory, University of Washington, Seat- tle, Washington. P. aeruginosa were grown on MacConkey's agar plates. P. aeruginosa strains were harvested from the surface of plates, and suspended in PBS to yield an optical density of 0.2 at 600nm. This suspension (10 s cfu. ml-i) was diluted in PBS to yield the desired inoculum.

C. Radioisotopes 14C-glucose (specific activity 12.9 GBq.mmo1-1) and 3H-thymidine (specific ac-

tivity 247.9 GBq. mmol-l) were obtained from New England Nuclear, Billerica, Mas- sachusetts.

D. Adherence assays L Double radiolabel. Chang cells were radiolabelled with 3H-thymidine on the 1st

day after seeding of the Leighton tubes by replacing nonradioactive ATCC CRCM-30 media with that containing 3H-thymidine at 3 #Ci-m1-1. When the cells were 40°7o confluent (8th day), they were challenged with radiolabelled bacteria. H. influenzae were radiolabelled by inoculating a single colony into 1 ml glucose-deficient Catlin's broth containing 100 #Ci of 14C-glucose. After overnight incubation at 37 °C, the bacteria were pelleted by centrifugation for 5 min at 15000 × g, resuspended in I ml PBS containing 0.1°70 gelatin (PBS-G) and again pelleted. =106 cfu (15068 dpm) were inoculated into each well of cell monolayer and incubated for 1 h at 37 °C with gentle agitation. The plastic slides containing the monolayers were removed from the Leighton tubes and washed five times by gentle agitation in 5 ml of PBS. Chang ceils and the associated bacteria were then removed from the slide by dripping 0.1 ml hya- mine hydrochloride (New England Nuclear) over the surface into scintillation vials. Glacial acetic acid (0.1 ml) was added to each vial, followed by 18 ml of Aquasol II (NEN). Contained 14C and 3H radioactivity was determined in a Packard scintillation counter with quench correction based on external standards. The specific radioactivity of the Chang cells was determined from the contained 3H and the number of cells (counted in a hemacytometer) released by trypsin. 14C-radiolabelled bacteria were diluted in PBS-G and plated on sBHI agar and the specific radioactivity calculated. Adherence was expressed as number of bacteria adherent. 100 Chang cells-1 calculat- ed from the measurements of radioactivity.

2. Kinetic assay. Respiratory epithelial monolayers, with or without the explant, in a Rose chamber were perfused with minimal Eagle's medium containing 1°70 gelatin at 6 ml. h-l . The bacterial strains to be studied (102 -103 cfu in 2 ml) were introduced into the intake port and the perfusion continued. Effluent from the Rose chamber was collected and 0.2-ml aliquots spread on the appropriate media, incubated overnight and the bacterial density determined: aliquots are cultured at 10-min intervals for 1-2 h. If there is no bacterial cell death, growth or adherence to the chamber or its contents, bacterial washout should be a simple exponential function. The washout can be described by d = do e-~t where d is the bacterial density at any time (t) and d o is the density at time equal to zero. If the above assumptions are true (i.e., no growth,

269

cell death or adherence), then the rate of washout should equal the perfusion rate. By perfusing the chamber with media that does not support measurable growth, and un- der conditions that maintain viability, the difference in rate of washout should reflect bacterial adherence. By comparing the washout of Rose chambers with and without eukaryotic cells, the adherence to mammalian cells can be assessed.

We compared the adherence of the various strains by calculating the mean time resi- dence (MRT) of the inoculum in the Rose chamber. The MRT was calculated by first determining the area under the bacterial density vs. time curve (AUC) extrapolated to infinity, using the trapezoidal rule. The area under the first moment curve (AUMC); i.e., the area under the product of bacteria density and time plot with respect to time was also obtained by the trapezoidal rule. The MRT [18] is calculated from the relation- ship: MRT = AUMC. AUC-1.

If the bacterial species can grow in the tissue culture media (which can be determined in separate experiments), the calculation of MRT remains the same and is still valid since the object here is to compare the MRT in the presence and the absence of eu- karyotic cells. That is, extraceUular growth of bacteria, if any, will be assumed to be equal in the absence and the presence of eukaryotic cells and can be determined by perfusing Rose chambers without eukaryotic cells.

3. Visual assessment. Buccal epithelial cells were obtained by scraping the inner cheek of normal adult humans, adult M. nemestrina or 6-wk-old male Sprague-Dawley rats with a wooden tongue blade. The cells were suspended in PBS, vigorously vortexed and pelleted by centrifugation at 15 000 × g for 10 min: this process was repeated three times. An aliquot was removed and the density determined with a hematocytometer. Chang cells were also harvested in suspension. The cellular density was adjusted to 105 cells, ml-1 by the addition of PBS and 108 cfu of H. influenzae added. After incu- bation at 37 °C for 60 min, the mixture was stained with methylene blue and the num- ber of adherent bacteria to 100 cells counted and the percentage of cells without detect- able bacteria adherent to their surface also determined.

E. Viability assessment

1. Trypan Blue exclusion. At selected time points after inoculation of bacteria into the Rose chamber, gelatin-coated glass slides with the respiratory epithelial tissue were removed, washed three times in PBS and the respiratory cells were removed by incubat- ing the slide for 5 min in 0.25°7o trypsin in 1 mM EDTA. The cell suspension was washed twice by pelleting and resuspending in PBS. The cells were resuspended in nor- mal saline containing 0.025 °7o Trypan Blue incubated at room temperature for 5 min and examined microscopically at 100x in a hematocytometer. 200 cells were examined and the number of staining was recorded and the percentage calculated.

2. 51Or release. Respiratory epithelial ceils or Chang cells were seeded onto 96 well microtiter plates and grown for 6 days at 37°C in 5°70 CO2. To each well, 10 tLCi of SlCr was added and the cells incubated at 37 °C overnight. The cells were washed three times with PBS and inoculated with the desired concentration of H. influenzae

270

or P. aeruginosa. At selected times, 75 #1 of culture supernatant was removed and the 51Cr content quantitated. 10007o lysis was achieved by resuspending the cells in 1070 tri- ton in water at room temperature: lysis is immediate when assessed visually. All sam- ples were counted within 60 min of being resuspended in triton. When performed in this manner, lysed cells are not differentiated from those detached. Viability was de- fined as the reciprocal of the percent lysis.

3. Cell metabolism. Respiratory epithelial cells were harvested from the explant cultures and seeded onto 96-wk microtiter plates and grown for 8 days at 37 °C in 507o CO 2. At the times indicated, with and without added bacteria, 10 #l of MTT (3-(4,5-dimethylthiazol-2 yl)-2,5-diphenyl tetrazolium bromide] (5 m g .m l - I PBS) was added and the incubation continued for 5 h. The purple-blue reduced MTT crys- tals could be seen within respiratory epithelial cells. Acid-isopropanol (100 #l of 0.04 N HCI in isopropanol) was added to solubilize the reduced MTT and the absorbance of each well at 570 nm determined with a Dynatech MR 580 plate reader, using 630 nm as a reference wavelength. Bacterial contribution to MTT reduction was assessed by adding the identical inoculum to separate wells and determining A570 nm" The absorb- ance at 570 nm at zero time was assumed to represent 10007o viability while no MTT reduction was assumed to represent no viable cells. The decrease in absorbance at 570 nm was used to calculate percent viability. This method is an adaptation of the cytotoxicity assay of Mosmann [19].

F. Hemagglutination titer Adult human type 0 erythrocytes were collected in heparin (1 U.m1-1) diluted

20-fold with PBS and centrifuged 10 min at 900 × g. The supernatant was decanted, the cells resuspended in a volume of PBS to yield a 3°7o suspension. Erythrocytes were washed by centrifuging at 1000 × g for 10 min at room temperature and decanting the supernatant. They were resuspended in the original volume of PBS and again cen- trifuged. This process was repeated four times prior to use in the hemagglutination (HA) assay. Bacteria were harvested from the surface of solid media and suspended in PBS to yield an absorbance of 0.6 at 600 nm. Serial two-fold dilutions of the bacteri- al suspension in PBS (50 #1) were added to 50/~1 of the 307o erythrocyte suspension on glass slides. After gentle agitation at room temperature for 15 min in a humidified environment, the HA titer was scored as the highest bacterial dilution yielding visible agglutination. A titer < 1:2 indicates there was no detectable agglutination at the lowest dilution of bacterial cells.

G. Cellular invasion The potential for H. influenzae inoculated into the Rose chamber to invade eu-

karyotic ceils was assessed in the following manner. After inoculation, the Rose cham- ber was perfused for _> 2 h. At the end of perfusion, the apparatus was disassembled and the tissue culture medium quantitatively cultured. The eukaryotic cells were then harvested with trypsin and the suspension vortexed with Eagle's medium and cen- trifuged at !000 × g for 10 min: the ceils were resuspended in the same volume of Eagle's medium and centrifuged twice. The supernatant was discarded, the cell suspension

271

serially diluted and plated on sBHI agar. These colonies are identified as cell- associated bacteria. Alternatively, the cell suspension was treated with gentamicin at 50/zg.m1-1 and incubated for 30 min at 37 oC. After washing three times, the cells were lysed by suspension in 1070 triton X-100. The lysate was serially diluted in PBS and plated on sBHI agar to determine intracellular bacterial density.

H. Statistical analysis The significance of differences between the means of replicate experiments with

each bacterial species and sources of respiratory epithelium were evaluated by means of a paired t test and by ANOVA, both derived with the BMDP statistical package for minicomputers [20].

Results

A. Eukaryote cytotoxicity We observed that the high inoculum of H. influenzae used in the standard double

label and visual assays appeared to be cytotoxic for respiratory epithelial cells. We, therefore, studied the effect of the size of the/-/, influenzae inoculum on viability of respiratory epithelial cells and Chang cells. Fig. 1 depicts the effect of increasing H. influenzae density on viability of Chang cells by Trypan Blue exclusion and 51Cr re- lease. With increasing duration of incubation as assessed by Trypan Blue exclusion or by 51Cr release using inoculum _> 106 cfu (those commonly used in conventional adherence assays), there is a decrease in a 20070 viability over 2 h (Fig. 1). However, we found that the 51Cr labelling of respiratory epithelial cells was low: 3-10 dpm above background was released by lysis of all cells with 1070 triton. The low specific activity of the cells (= 4 cpm. eukaryote cell-~) precludes reliance on this assay. Thus, we could not confirm that high inoculae of H. influenzae was cytotoxic for respiratory epithelium using 51Cr release. Therefore, in addition to Trypan Blue exclusion we used the ability of the cells to reduce tetrazolium as an index of cytotoxicity. Fig. 2 depicts the time-dependent and inoculum-dependent loss of viability of primate respiratory epithelial cells after exposure to H. influenzae using this assay. After 2 h of incubation

V i a b i l i t y of c h a n g c o n j u n c t i v a l cel ls a f t e r i n c u b a t i o n w i t h H. i n f l uenzae f -

9O ~ 80

6o " - . 5 0

8 40 • 6 - - A lO 8 z 3 0 . 5,c~ ~ •

2 0 o - - - o 104 ~ 1 0 - e - - - e lo 6 ",~

- A - - - a lO 8

q ~ 4 & T i m e ( h )

Fig. I. E f f e c t o f H . influenzaeinoculumsizeonviabilityofChangcellsasassessedbyTrypanBlueexclu- s ion (solid line) an d 51Cr release (dashed line). Inocu la were 104 ( 0 ), 106 ( • ) and 108 cfu ( A ).

272

Fig. 2.

1ooi 901

7O o • ~ 6 0

5O

~ 4o e 30

2O 10

0

Viabil i ty of m o n k e y tracheal cells a f t e r i n c u b a t i o n w i t h H. J n f l u e n z a e F i m -

-" " - - 7 7 - . . . . $ Xr ypon blue ~A

O - - O L O 4 o - - 1 1 1 o 6 A - - ~ 10 8

MTT o---o 1o 4

" &---A Io e

"rime (h)

Effect of 1-1. influenzae b inoculum density on viability of primate respiratory epithelium assessed over time by MTT reduction (dashed line) and Trypan Blue exclusion.

with 104 or 108 cfu of H. influenzae, viability had decreased by 20°70. This is similar to the inoculum-dependent cytotoxicity seen with Chang conjunctival cells (compare Figs. 1 and 2). After 6 h of exposure of the ceils to 108 cfu of type b H. influenzae,

½ of the ceils are not generating reduced nicotinamide adenine dinucleotide. These data indicate that bacterial adherence should be assessed with low inocula and for ex- posure times < 2 h.

Inoculating large numbers of bacteria may indicate adherence to damaged or dying as well as viable cells. We, therefore, sought to define the ability of a kinetic assay to identify bacteria with increased adherence.

B. H. influenzae adherence Fimbriated type b H. influenzae are more adherent to human and monkey but not

rat buccal and pharyngeal epithelial cells in comparison to isolates lacking surface tim- briae [9, 10]. Table 1 shows the increased adherence to primate cells confirming the species specificity of buccal epithelial cell adherence. To determine whether increased adherence in the conventional assay was detectable in a kinetic assay, we determined the rate of washout of isogenic fimbriated and nonfimbriated H. influenzae from Rose chambers containing monkey respiratory epithelium. Shown in Fig. 3, is the washout

T A B L E 1

A D H E R E N C E O F T Y P E B H. I N F L U E N Z A E T O E U K A R Y O T I C C E L L S

Strain Adherence to B E C ( n . c e l l - 1 )

Source of B E C

Human Monkey Rat

E l a F i m - 0 .47 _+ 0 .38 0 .60 _+ 0 .46 0 .08 + 0 .06 R1369 F i m + 8 .0 _-4- 0 .82* 12.66 + 2.80* 0 .07 _+ 0 .07

+ SD is given. * F i m + is > F i m - a t P = 0 .023 , A N O V A .

273

H. influenzae b adherence to primate respiratory epithelium

~ . 6

8 5q

~ 4 u.

J 2 O ~ -

0 Elo 0 1 • R 1369

06 ..... 1~ ~o 3b ~o ~ T i m e (min)

Fig. 3. Elimination of Fim + (R1369) and F im- (Ela) H. influenzae b from Rose chambers perfused with Eagle's minimal media at 6 ml .min -1 . Inocula were 1240 cfu of Ela and 860 cfu of R1369, each in 2 ml.

curve of Fim ÷ and F im- type b H. influenzae from Rose chambers containing respiratory epithelial explants. If the MRT is calculated from this data, strain R1369 (Fim ÷) is more adherent; an average of 18.5 vs. 12.3 min for strain Ela. Inoculation of perfused Rose chambers not containing respiratory epithelium permits assessment of bacterial adherence to the glass and plastic components. Table 2 depicts the MRT of these same two strains of H. influenzae in perfused Rose chambers, in chambers with rabbit respiratory epithelial explants and in chambers containing subhuman pri- mate respiratory epithelial explants. The data indicates that Fim + H. influenzae b are more adherent to chamber components but the presence of primate epithelium addi- tionally increases MRT. An increased MRT is seen with rabbit respiratory epithelium but the Fim + strain is not statistically different from the F im- isolate (P>0.2). The longer MRT of R1369 Fim + with primate respiratory epithelium correlates with the increased adherence to human buccal epithelial cells (BEC) (Table 1). Adherence of strain Ela F im- to BEC was 0.47 + 0.38 bacteria.BEC -l while with the isogenic derivative R1369 Fim ÷ it was 8.0 + 8.2 bacteria.BEC -1. With Ela F im- 14 + 12°70 of the BEC had detectable bacteria: with R1369 Fim ÷ 82 + 807o of the BEC had ad- herent bacteria. Using the double-label technique, we found that neither R1369 Fim ÷

TABLE 2

ADHERENCE OF TYPE B H. I N F L U E N Z A E TO RESPIRATORY EPITHELIUM

Strain MRT (min)

Epithelial cell species

Rabbit (8) Monkey (7) None (3)

E la F im- 18.06 :t: 4.18 12.29 _+ 2.19 11.39 4-_ 3.30 R1369 Fim + 17.69 + 4.06 18.51 ± 1.97" 13.73 4- 2.58

(n) indicates number of replicate experiments. + SD is given.

* Fim + is > F i r e - at P = 0.023, ANOVA. ** Fim + has a greater MRT in presence of monkey epithelium, P = 0.09.

274

nor Ela Fim- was measurably adherent to Chang cells (Table 3). This result was con- firmed by visual assessment (Table 3).

C. Cellular invasion As we previously noted, Fim ÷ H. influenzae b (R1369) is more adherent to a blank

cassette but there is a eukaryote cell-dependent increase in bacterial density in the chamber, the number of cell-associated organisms and the number of intracellular bac- teria ( P < 0.009). The Fim- strain, Ela, is also more adherent to the chamber when eukaryotic cells are present but the difference is not as great as seen with the Fim + strain and is not statistically significant. The increased adherence of the Fim + strain appears to result in increased invasion of eukaryotic cells (Fig. 4).

D. Adherence of Pseudomonas To determine if the kinetic assay would allow determination of adherence of other

respiratory bacteria to airway epithelial cells, we studied a piliated P. aeruginosa and the isogenic strain with the pili structural gene inactivated. In addition, the adherence of an afimbriate P. cepacia and a fimbriated derivative was studied.

With Pseudomonas species (Table 4), we found that the fimbriated P. cepacia strains

T A B L E 3

A D H E R E N C E O F T Y P E B H. INFL U E N Z A E T O C H A N G C O N J U N C T I V A L C E L L S B Y D O U B L E - R A D I O L A B E L T E C H N I Q U E

Strain HA titer Bacteria- cell - l

Radiolabel assay Visual assay

E l a F i m - < 1 : 2 5 .2 x 10 - 4 0 .02 _ 0 .03 R1369 F i m + 1 : 32 9.3 x 10 - 4 0 .05 + 0 .03

HA titer performed as described in Materials and Methods.

O.OOe

- E O.OO~

= _~ 0 . 0 0 4 .

= o o oo3- U . E "6 ~ O.OO2- .o_ U

O.OO1 -

0.00(3

Bacterial adherence to rabbit tracheal cells

/'1 PIostic support I:~ Ceff assosciated I~ Lysed cells O Glass support

R1369 R1369 Ela Ela with without with without

(Rabbit t racheal cells)

Fig. 4. Eukaryote invasion was assessed by culturing Rose chamber components after 60 min perfusion at 6 m l - m i n -1 with and without respiratory epithelial cells. Those adherent to cassette, respiratory epitheli- al ceils and those intracellular (gentamicin-resistant) are depicted. Bacteria, 14200 cfu of strain Ela, were

in contact with the eukaryotic cells for 180 min at 3 7 ° C .

275

TABLE 4

HEMAGGLUTINATING ACTIVITY AND ADHERENCE OF PSEUDOMONAS TO MONKEY RESPIRATORY EPITHELIAL CELLS

Strain HA titer MRT (min)

P. aeruginosa Fire- < 1 : 1 15.40 + 5.53 [3] P. aeruginosa Fim + < 1 : 1 12.69 + 1.59 [3]

P. cepacia Fire- < 1 : 2 13.57 _+ 2.04 [4] P. cepacia Fim + 1 : 8 16.33 + 2.10 [6]

Mean MRT + SD is depicted. [ ] indicates number of replicate experiments. Fim + significantly greater than Fire- at P< 0.03.

selected by erythrocyte adsorpt ion had hemagglut inat ion activity and increased adher- ence to rabbit respiratory epithelium as reflected by a longer MRT. In contrast , the f imbriated P. aeruginosa did not have detectable H A activity with h u m a n erythrocytes and did not have a statistically longer MRT with rabbit respiratory epithelium in com- par ison to the strain with the gene encoding the fimbrial subunit inactivated (Table 4). We also found that high densities o f P. aeruginosa (>_ 108 cfu) were cytotoxic to rabbit respiratory epithelium in a t ime-dependent fashion as measured by Trypan Blue exclu- sion (Fig. 5). After 6 h o f incubat ion with 106 P. cepacia F i m - (PC 109), the monolayer was examined by scanning electron microscopy and compared to an uninoculated monolayer (Fig. 6). Clumps o f bacteria can be seen adhering to extruded ciliated and nonciliated cells.

Discussion

During the assessment o f the adherence o f H. influenzae b to cultured respiratory epithelial cells, we observed apparent cytotoxicity: cells became round and desquamat- ed. We then sought to directly test for cytotoxicity and, if present, devise an alternative

Fig. 5.

100

70

60

50

30 T~y~n blue

20 O--O 104 I 0 " O--e 108

I 0

Viability o i rqbbit tracheal cells after incubation with F~ aeruginosa

Time (h)

Effect of P. aeruginosa PAK inoculum density on viability of rabbit respiratory.epitheliai cell ex- plants with time assessed by Trypan Blue exclusion.

276

a

Fig. 6. (A) Scanning electron photomicrograph of respiratory epithelial explant after inoculation with cul- ture media. Magnification is 8000 ×. (B) Parallel explant after inoculation with 4.3 × 10 7 cfu PC 109 P. cepacia for 6 h at 37 °C. Bacteria are associated with extruding ciliated and nonciliated cells (arrows). Mag-

nification is 12000 ×.

method which would permit testing of the adherence of low numbers of bacteria. We found inoculum and time-dependent cytotoxic effect of H. influenzae and P. cepacia on Chang conjunctival cells and primate respiratory epithelial monolayers. From these experiments, we concluded that testing of adherence with > 106 cfu should be per- formed in < 2 h if target cell cytotoxicity is to be avoided.

An additional disadvantage of using inocula > 106 cfu is that invasion could not be tested within the same experiment. When human respiratory epithelium is tested, it is ideal that adherence and invasion be tested with the scarce tissue. In vivo bacterial adherence overcomes shear forces generated by the mucus blanket (mucociliary clear- ance) which would remove the organisms. We, therefore, devised a kinetic method which would permit testing bacterial adhesion in a flowing system and invasion within the same experiment.

A kinetic means of assessing bacterial adherence to eukaryotic cells in Rose cham-

ia

277

bers has several advantages over static techniques. The inoculum can be as low as sever- al 100 bacteria which maintains the normal morphology of the target tissue and adher- ence capacity is tested in a flowing system as it is in vivo. The MRT of a bacterium in the Rose chamber eliminates observer bias and tests all of the eukaryotic cells pres- ent. Inoculation of low numbers of bacteria can mimic the inoculum thought to occur in vivo and avoids cell damage from LPS and secreted bacterial toxins. High densities of H. influenzae have previously been shown to produce degeneration of hamster tracheal epithelium in organ culture. This effect is thought to be due to lipopolysaccha- ride [21]. Other bacteria, such as P. aeruginosa, are known to secrete protease, lipase and exotoxin A, all of which can decrease the viability of eukaryotic epithelium. Ex- amination of the cells in the explant with the scanning electron microscopy can identify target cell preference by adhering bacteria. However, with low noncytotoxic inocula, the procedure is time consuming as the bacteria are widely dispersed.

Disadvantages of this method include the need for tissue culture facilities, availabili- ty of the respiratory tissue of the species of interest and expertise in establishing viable respiratory epithelial cell monolayers. Quantitation of the percentage of epithelial cells

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with adherent bacteria and the average number of bacteria adherent to each cell is not conveniently performed with this assay. Another disadvantage of this method is the intrinsic variability in the enumeration of small numbers of bacteria. Bacteria, like all particles in low concentration, follow a Poisson distribution. To obtain reliable data on the bacterial density vs. time in the effluent using low inocula, the experiment has to be repeated several times. Increasing the bacterial inoculum increases the confidence of the estimation of bacterial density in a single experiment but must be balanced against potential cytotoxicity occurring during exposure to large numbers of bacteria for incubation times > 2 h. With tissue exposure times < 1 h, higher numbers of bac- teria can be reliably tested for adherence.

Increasing the inoculum to l04 cfu leads to more reproducible washout curves and, correspondingly, smaller variance in the MRT. With subhuman primate respiratory epithelium, the MRT ranges from 16.91 to 21.45 min with inoculum between 43 and 9020 cfu of H. influenzae b Fim +. With the Fire- H. influenzae b, there is a tendency toward increasing the MRT as the inoculum is increased: 10.30 min with 155 cfu and 16.42 min at 9550 cfu, for example. This data suggests saturation of a low-number high-affinity receptor for Fire + H. influenzae. The minimal increase in adherence of Fim + H. influenzae with increasing inoculae suggests a low-affinity receptor present in abundance in respiratory epithelium which is saturated at low bacterial density.

We also examined flow rates between 0.6 to 60 ml. h -1 with Fim ÷ and Fim- H. in- fluenzae b with inocula of 102 -103 cfui In general, the MRT decreases as the flow rate increases, up to a rate of 30 ml. h -~. At flow rates exceeding that value, effluent sampling must be at 30-s intervals to accurately describe bacterial washout from the chamber; a procedure performed with difficulty. It appears that MRT does not de- crease further when the flow rate exceeds 30 ml. h-1.

Greater adherence of fimbriated type a H. influenzae to respiratory epithelial cells was demonstrated with the kinetic assay. The longer MRT of the Fim ÷ isolated was also reflected in greater adherence to human BEC, as has been reported, but not to Chang conjunctival cells. Since H. influenzae can cause conjunctivitis, it is surprising that these cells did not provide a ligand for adherence. However, most conjunctival H. influenzae isolates do not possess a capsule and are biotype III [22]. In contrast, invasive H. influenzae usually possess type b capsule and are biotype I.

P.. aeruginosa PAK can adhere to BEC and the adherence is decreased by preincuba- tion with intact pili [23]. However, it is not clear that pili are the primary ligand. Doig et al. [24] suggest that there are two ligands on the surface of P.. aeruginosa for tryp- sinized BEC. Others have suggested that the tracheal epithelium must be injured for P. aeruginosa to adhere or that the organisms bind to tracheal mucin [25]. Recent data suggest that asiaylgangliosides are the eukaryotic receptor and these moieties are not normally surface-exposed [26]. Other data comparing adherence of isogenic P. aeru- ginosa lacking or possessing fimbriae are not available. In contrast to P. aeruginosa, the fimbriated P.. cepacia had a longer MRT with rabbit respiratory epithelium than the Fim- parent. Since fimbriation is selected for by hemagglutination with human erythrocytes, the ligand on the surface of rabbit tracheal epithelium may be structurally similar to that on erythrocytes.

We conclude that the kinetic assay has certain advantages over existing methods, permitting evaluation of adherence of small numbers of viable bacteria with the natu-

279

ral t a rge t t issue. T h e m e t h o d c o m p a r e d f avo rab ly wi th o t h e r t e c h n i q u e s a n d c o n f i r m e d

species spec i f i c i ty wi th H. influenzae. Bac te r i a l s t ra ins f o u n d to have inc reased adher - ence in s t a n d a r d assays h a d an i nc rea sed MRT.

Acknowledgements

T h i s w o r k was s u p p o r t e d in pa r t by a g r an t f r o m t h e N a t i o n a l Ins t i tu te o f A l l e r g y a n d

I n f e c t i o u s Disease ( A I 20625) a n d the Cys t ic F ib ros i s F o u n d a t i o n reg iona l deve lop- m e n t p r o g r a m .

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