Role of Mechanical Injury on Airway Surface in the Pathogenesis of Pseudomonas aeruginosa 1 ,2
TSUNEKO YAMAGUCHI and HOZUMI YAMADA Introduction
In general, gram-negative bacteria are frequently cultured from patients with structural epithelial damage, such as results from prolonged endotracheal intubation, acid aspiration, and tracheostomy (1-4). In these colonizing bacteria, Pseudomonas aeruginosa is clinicallythe most important colonizer in the respiratory tract for its low susceptibility to antibiotics. Why does R aeruginosa colonize in the respiratory tract? In an attempt to answer this question, Ramphal and colleagues (5) suggested the following for consideration: (1) R aeruginosa has a tropism for the respiratory tract; (2) there must be a defect in physical clearance; and (3) there must be a defect in immune clearance. These three mechanisms must playa role in the colonization of this organism on airway surface. Although R aeruginosa has a tropism toward airway surface (6-8), it has been clinically recognized that colonization or infection by this organism occurs when bronchial mucosa is injured (4). And, in vitro, it has been revealedthat acid-injury on airway surface enhanced attachment of this organism (9). In clinical terms, however, most tracheal damages are mechanical injuries, such as caused by intubation or suction tubes, especially when patients are kept under management with intubation. To the best of our knowledge, however, there have been no reports dealing with the binding sites for R aeruginosa induced by mechanical injury on airway surface. In this report, we shall demonstrate an enhanced attachment of R aeruginosa by airway surface damage and an enhanced growth of this organism after binding to injured sites using an in vitro experiment system of rat trachea. Weshall also carry out a partial characterization of the receptor on brush-injured tracheal surface for this organism. Methods Bacterial Preparation Three strains of piliated, nonmucoid type of R aeruginosa (ATCC27853,SAGA 3606, and
SUMMARY In this study, bacterial attachment to rat tracheal surface was measured using three nonmucold strains of Pssudomonas asruglnosa and bacterial growth after binding to tracheal surface was also teste\j. Brush Injury on tracheal surface significantly Increased bacterial attachment (1,190to 1,600%); bacteria binding to brush-Injured sites grew more rapidly than either nonbinding bacteria or those on Intact trachea. A partial characterization of the binding sites for P. asruglnosa on either Intact or injured tracheal surface also was performed. Treatment of Injured tracheal surface with metaperlodate significantly Inhibited attachment of P. asruginosa, but trypsin treatment did not. In contrast, neither reagent had any effects on bacterial attachment to Intact tracheal surface. These resuIts suggest that brush Injury on tracheal surface produces new binding sites as a receptor for P. asruginosa, and that this receptor has carbohydrates as Important components and that It Is not a protein receptor. In addition, In order to determine what the dominant sugar of this receptor was, we tested the Inhibition of bacterial attachment with monosaccharide, neuraminldase, and lectin. Treatment of bacteria with N·acetylneuramlnlc acid (NANA) dramatically In· hibited bacterial attachment to injured trachea. However, NANA also inhibited the growth of this organism. Moreover, neither neuraminidase nor lectin data suggested that the dominant sugar of the receptor was NANA. Our data go so far as to confirm that the major component of the receptor of nonmucoid strains of P. asruglnosa on brush·injured trachea Is carbohydrates; It Is stili unclear what kind of sugar Is the dominant component of the receptor. We have concluded that mechanical injury, such as caused by brushing, Induces a promoting effect on attachment and growth of P. aeruginosa, and that this effect is attributed to production of the receptor that Is not available with Intact trachea. Such a tropism of P. aeruglnosa to airway surface Induced by mechanical injury Is likely to play an Important role in the colonization and Infection of this organism. AM REV RESPIR DIS 1991; 144:1147-1152
SAGA 3760) were used in this study. SAGA 3606 and SAGA 3760 strains were isolated from sputum. Each organism was grown in Trypticase soy broth (Becton Corp., Cockeysville, MD) for 16 h at 37° C, washed three times in sterile saline, resuspended in sterile saline, and then adjusted to an optical density of 0.07 for each strain at 650 nm in a spectrophotometer. These concentrations of bacterial solution contained approximately 3 x 107 colony-forming units (cfu)/ml.
Rat Tracheal Preparation A total of 192male Donryu rats, 11 to 14 wk old, were obtained from Kyudoh Corp. (Fukuoka, Japan). Animals were killed by intraperitoneal injection of 500 to 800 mg/kg pentobarbital, and then the trachea was excised and cut to 1.5 to 2.0 em in length. In the injured trachea group, tracheal surface was brushed with a small bronchoscopic brush (BC-IO; Olympus Corp., Tokyo, Japan). After brushing, the tracheal lumen was washed with 2 ml sterile saline. We then observed the turbidity caused by desquamative cells and tissue debris in each solution washed. Samples having the same degree of turbidity demonstrated a similar severity of histologic injury. Microscopically, specimens of brushinjured rat trachea presented severe damages
of epithelial cells; most ciliated cells were lost and submucosal tissues were naked.
Bacterial Attachment 'Testing In this study, we modified the adherence assay reported by Marcus and coworkers (10, 11), who carried out a quantitative analysis of bacterial adherence using a culture method. In the bacterial attachment testing, three non mucoid strains of R aeruginosa (ATCC 27853, SAGA 3606, and SAGA 3760) were used and five animals were killed in each group. Excised rat trachea was connected to small tubes 1.5mm in diameter. Tracheal surface was then rinsed with sterile saline, and a bacterial solution adjusted to the concentration of 3 x 107 cfu/ml was instilled into the tracheal lumen. Then the tracheal preparation wasincubated for 1hat 37° C in Hanks'
(Received in original form May 30, 1990 and in revised form May 1, 1991) I From the Department of Internal Medicine, Saga Medical School, Saga, Japan. 2 Correspondence and requests for reprints should be addressed to Dr. Hozumi Yamada, Department of Internal Medicine, Saga Medical School, I-I, Nabeshima 5-chome, Saga 849, Japan.
1147
1148
solution. In each experiment, a small volume of Hanks' solution, in which the tracheal preparation had been incubated for 1 h, was cultured to confirm no leakage of bacterial solution from the tracheal lumen. After a 1-h incubation, the tracheal lumen was rinsed with 120 ml sterile saline to wash out nonbinding bacteria. Byscanning electron microscopy,we ascertained that bacteria directly attached to intratracheal surface. For a quantitative analysis of binding bacteria, the tracheal preparation was homogenized in 2 ml of sterile saline. After huge materials were removed by meshes, a volume of 50 III of each homogenized solution was spread on a modified drigalski agar plate (BTB;Eiken Corp., Tokyo, Japan) using the Spiral System (Spiral System Inc., Cincinnati, OH). After overnight incubation, bacterial colonies on plates were counted with a Laser Bacteria Colony Counter (Spiral System). A quantitative analysis using a combination of both types of equipment presented reproducible data. Bacteria binding to tracheal surface were represented as a number of bacteria per square millimeter of tracheal surface.
Partial Characterization of Binding Sites The following reagents were obtained from Sigma Chemical Corp. (St. Louis, MO): trypsin, sodium metaperiodate, D - ( +)- mannose (mannose), L- (- ) - fucose (fucose), N-acetylneuraminic acid (NANA), N-acetylgalactosamine (GaINAc), N-acetylglucosamine (GIcNAc), Vibrio cholerae neuraminidase type III, Clostridium perfringens neuraminidase type V,Arachis hypogaealectin, Pisum sativum lectin type III, and Limuluspolyphemus lectin. In the characterization experiments, we used three nonmucoid strains of R aeruginosa (ATCC27853, SAGA 3606,and SAGA 3760)and killed three animals in each group. In order to perform a partial characterization of the binding sites for R aeruginosa on either intact or brush-injured tracheal surface, we used metaperiodate, which oxidizes carbohydrates, and trypsin, which destroys protein components. Rat tracheal surface was rinsed with 20 ml sterilesaline and then treated with 90 mM sodium metaperiodate (pH 5.0) for 40 min at room temperature or with 20 ug/ml trypsin for 15min at 25 0 C. In the control group, we used sterile saline instead of either reagent. After treatment with a reagent, the tracheal surface was rinsed with 60 ml sterile saline to wash out the reagent completely. The control tracheal preparations also were rinsed with sterile saline before exposure to bacteria. Bacterial attachment testing was then carried out as described previously. The results were represented as a number of bacteria per square millimeter 0 f tracheal surface. In addition, weused monosaccharides, neuraminidases, and lectins to perform a further characterization of the binding sites for R aeruginosa on brush-injured tracheal surface. In the monosaccharide experiments, R aeruginosa was incubated for 1 h at 370 C with
YAMAGUCHI AND YAMADA
various sugars: mannose (100 mM), fucose (100 mM), GIcNAc (100 mM), GalNAc (100 mM), and NANA (1 mM). In the control group, bacteria were incubated in sterile saline for 1 hat 37 0 C. After incubation, bacteria were washed three times in sterile saline and then bacterial attachment testing was carried out with injured tracheal preparations. The result of bacterial attachment was represented as a ratio(%) to control. Moreover, the growth of R aeruginosa treated with each monosaccharide was measured as the optical density changed while organisms were incubated in Hanks' solution containing 10% fetal bovine serum(FBS) at 370 C. In the neuraminidase and lectin experiments, injured tracheal surface was treated with 0.2 U neuraminidase type III in 0.15M NaCI and 4 mM CaCl 2 (pH 5.5) or 2.0 U neuraminidase type V in 50 mM sodium acetate (pH 4.9) for 40 min at 37 0 C in Hanks' solution; it also was treated with 100 ug/ml of A. hypogaea lectin, R sativum lectin type III, and L. polyphemus lectin for 40 min at 25 0 C. In both experiments, we used sterile saline instead of a reagent in the control group. After treatment with either reagent, the tracheal surface was rinsed with 60 ml sterile saline to wash out the reagent. The control tracheal preparations also were rinsed with sterile saline before bacterial exposure. Bacterial attachment testing was then carried out. The result of bacterial attachment was represented as a ratio (%) to control.
Bacterial Growth Testing For the growth testing of bacteria binding to rat tracheal surface, three strains of nonmucoid R aeruginosa (ATCC27853, SAGA 3606, and SAGA 3760)wereused and three animals were killed in each group. The tracheal preparation was exposed to bacterial solution, which was adjusted to an optical density of 0.20 in the intact trachea group or to an optical density of 0.02 in the brush-injured trachea group. After a 1-hexposure, the tracheal lumen was rinsed with sterile saline, and then the tracheal preparation was cut into two pieces; one piece was homogenized immediately for the counting of binding bacteria and the other was incubated for 90 min at 37 0 C in 1 ml Hanks' solution containing 10% FBS to observe bacterial growth after binding. After the 90-min incubation, the tracheal preparation was homogenized. The number of bacteria was counted with the same management as described above.The results wererepresented as a number of bacteria per mIlt of tracheal surface. On the other hand, the growth of nonbinding bacteria also was measured as control when a total number of approximately 1.0 x 104 cfu/O.l ml of each strain was incubated for 1 h at 37 0 C in 1 ml Hanks' solution including 10% FBS. The results were represented as numbers of bacteria per 0.1 ml. Statistical Analysis Each result represented the mean ± standard deviation (SD) of five animals for bacterial
attachment; the mean of three animals was used for the characterization of binding sites. Statistical analysis was performed by the unpaired Student's t test.
Results
Bacterial Attachment to Tracheal Surface Bacterial attachment to tracheal surface was tested using three strains of nonmucoid R aeruginosa. The results of this testing are shown in table 1. The numbers of bacteria binding to intact trachea were 4.4 ± 1.4 x 103 cfu/mm? for strain ATCC 27853, 4.4 ± 1.2 x 103 cfu/mmfor SAGA 3606, and 3.9 ± 1.7 x 103 cfu/mm? for SAGA 3760. The number of bacteria binding to tracheal surface significantly increased by brush-injury (ATCC 27853 = 1,190070 of intact, p < 0.01; SAGA 3606 = 1,290070 of intact, p < 0.01; SAGA 3760 = 1,600070 of intact, p < 0.01). Figure 1 shows the electron microscopic findings of bacterial attachment on intact tracheal surface (figure lA) and on brush-injured tracheal surface (figure IB). A few bacteria are found on cilia of intact epithelial cells (figure lA). In contrast, figure IB shows that most of the epithelial cells are lost and a huge number of bacteria are binding to naked submucosal connective tissue directly. Characterization of Binding Sites In order to perform a partial characterization of the binding sites on either intact or brush-injured tracheal surface, we tested the influence on bacterial attachment of metaperiodate, which oxidizes carbohydrates and trypsin, which destroys protein components. Effects of both reagents on bacterial attachment are shown in figure 2. Treatment with 90 mM metaperiodate significantly reduced the TABLE 1 EFFECTS OF BRUSH INJURY OF TRACHEAL SURFACE ON BACTERIAL ATIACHMENT TO RAT TRACHEA·
Nonmucoid Strains of P. aeruginosa ATCC 27853 SAGA 3606 SAGA 3760
No. of Bacteria Binding to Trachea (x 103 cfu/mm2) Intact
Brush-injured
4.4 ± 1.4 4.4 ± 1.2 3.9 ± 1.7
52.4 ± 11.2t 56.8 ± 15.9t 62.7 ± 11.8t
• Three nonmucoid strains of P. aeruginosa were tested and tracheal surface was injured with a bronchoscopic brush. Binding bacteria are represented as a number of bacteria per square millimeter of tracheal surface. Each result represents the mean ± SO of five animals. Statistical analysis was performed by the unpaired Student's t test.
t p < 0.01.
AIRWAY SURFACE DAMAGE AND PATHOGENESIS OF P. AERUGINOSA
1149
Fig. 1. Intact tracheal surface is covered with normal ciliated epithelial cells and a small number of P. aeruginosa (ATCC27853) are found on cilia (A). In contrast, brush-injured tracheal surface has completely lost ciliated epithelial cells and submucosal connective tissue is naked. Huge numbers of bacteria (ATCC 27853) have directly attached to submucosal connective tissue (B).
(AI
number of bacteria binding to brushinjured trachea (ATCC 27853, 19% of control; SAGA 3606, 26070 of control; SAGA 3760, 22% of control), but trypsin treatment had little effect on bacterial attachment (ATCC 27853, 109% of control; SAGA 3606, 92010 of control; SAGA 3760, 74% of control). In contrast, neither reagent had any effect on bacterial attachment to intact tracheal surface (metaperiodate: ATCC 27853, 110070 of control; SAGA 3606, 134% of control; SAGA 3760, 159% of control; trypsin: ATCC 27853, 86% of control; SAGA 3606, 77% of control; SAGA 3760, 141 % of control). These data reveal that the binding sites for R aeruginosa on injured tracheal surface are different from those on intact tracheal surface. It is concluded that brush injury on tracheal surface produces newbinding sites for R aeruginosa as a receptor, and that this receptor consists of carbo-
ATCC27853
5AGA3606
5AGA3760
1.0
0.8
Fig. 2. Effects of metaperiodate or trypsin on attachment of three nonmucoid strains of P. aeruginosa to either intact (A) or brush-injured (B ) trachea. Before bacterial exposure, rat trachea was treated with 90 mM metaperiodate (solid bars) or 20 lJ.9/ml of trypsin (hatched bars). In the control group, tracheal preparations were also treated with sterile saline (open bars) instead of either reagent. Bacteria (3 x 107 cfu/ml) were exposed to rat tracheal surface for 1 h at yo C. Binding bacteria (ordinate) represent the mean ± SD ofthree animals. Statistical analysis was performed by the unpaired Student's t test. Asterisks indicate significant values of difference compared with control (p < 0.01).
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1150
hydrates that are an important component and that it is not a protein receptor. For a further characterization of the receptors produced by brushing, we also tested the influence on bacterial attachment of monosaccharide, neuraminidase, and lectin (table 2). Mannose (100mM), fucose (100mM), GlcNAc (100mM), and GalNAc (100mM) had little effect on attachment of these nonmucoid strains of R aeruginosa to injured tracheal surface, but 1mM NANA significantly inhibited their attachment (ATCC 27853, 5070 of control; SAGA 3606, 10070 of control; SAGA 3760,3% of control). On the other hand, figure 3 shows the growth of R aeruginosa after treatment with each of the monosaccharides tested. Mannose, fucose, GlcNAc, and GalNAc had hardly any effect on the growth of R aeruginosa, but NANA dramatically inhibited the growth of this organism. The effects of two different types of neuraminidase on bacterial attachment are given in table 2. JI: cholerae neuraminidase type III (0.2 U) did not decrease bacterial attachment to injured trachea (ATCC 27853, 88% of control; SAGA 3606, 105% of control; SAGA 3760, 104% of control), nor did C perfringens neuraminidase type V (2 U) decrease bacterial attachment (ATCC 27853, 73070 of control; SAGA 3606, 115070 of control; SAGA 3760, 106% of control). The lectin data are also shown in table 2. Treatment of injured tracheal surface with lectins from A. hypogaea, (103 to 151070 of control), R sativum, (80 to 109% of control), and L. polyphemus (85 to 107% of control), did not inhibit attachment of these organisms.
Bacterial Growth Testing R aeruginosa binding to rat trachea was incubated for 90 min in Hanks' solution containing 10070 FBS. The numbers of binding bacteria before and after the 90min incubation are shown in figure 4. During the 90-min incubation, bacteria on intact trachea increased from 1.6 X 104 to 3.1 X 104 cfu/mm" (ATCC 27853), from 1.2 X 104 to 3.6 X 104 cfu/mm" (SAGA 3606), and from 1.2 X 104 to 3.1 X 104 cfu/mm? (SAGA 3760); those on brush-injured trachea increased from 0.8 X 104 to 4.2 X 104 cfu/mm-, from 1.2 x 104 to 8.6 X 104 cfu/mm-, and from 1.0 X 104 to 6.4 X 104 cfu/mm-, respectively. On the other hand, the growth of nonbinding bacteria also was measured as control in each strain and the result is also shown in figure 4. During the 90min incubation, nonbinding bacteria in-
YAMAGUCHI AND YAMADA
TABLE 2 EFFECTS OF MONOSACCHARIDE, NEURAMINIDASE, AND LECTIN ON ATIACHMENT OF P. AERUGINOSA TO BRUSH-INJURED TRACHEA' Attachment of P. aeruginosa to Brush-injured Trachea (%)
Reagents Monosaccharide Mannose, 100 mM Fucose, 100 mM GlcNAc, 100 mM GaINAc, 100 mM NANA, 1 mM Neuraminidase V. cho/erae, 0.2 U C. perfringens, 2 U Lectin A. hypogaea, 100 ~g/ml P. sa tivum , 100 ~g/ml L. polyphemus, 100 ~g/ml
ATCC 27853
SAGA 3606
SAGA 3760
98 115 76 105 5
86 124 85 92 10
114 90 101 97 3
88 73
105 115
104 106
151 109 107
132 80 85
103 93 89
Definition of abbreviations: GlcNAc = N-acetylglucosamine; GalNAC = N-acetylgalactosamine; NANA = N-acetylneuraminic acid; V. cholerae = Vibrio cholerae type III; C. perfringens = Clostridium perfringens type V; A. hypogaea = Arachis hypogaea; P. sativum = Pisum sativum type III; L. polyphemus = Limulus polyphemus. • In the monosaccharide testing, bacteria were incubated with each monosaccharide before exposure to injured trachea. In the neuraminidase and lectin testing, injured tracheal preparations were treated with neuraminidase or lectin before bacterial exposure. The results of bacterial attachment were represented as a ratio (%) to control using saline instead of a reagent. Each result represents the mean of three animals.
ATCC 27853
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Fig. 3. Growth of P. aeruginosa (ATCC 27853, SAGA 3606, and SAGA 3760) after treatment with various monosaccharides: mannose (100 mM), fucose (100 mM), GlcNAc (100 mM), GalNAc (100 mM), and NANA (1 mM). Control bacteria were treated with sterile saline instead of monosaccharides. Bacterial growth was measured according to how the optical density changed in Hanks' solution cdntaining 10% FBS while incubated at 37" C.
AIRWAY SURFACE DAMAGE AND PATHOGENESIS OF P. AERUGINOSA
non. Previous investigators reported that a tropism of the respiratory tract on the part of R aeruginosa plays an important 6 role in the development of infections binding (injured) 4 (6-8). Marcus and colleagues (10)exam_ ... binding (intact) ined the mechanism of attachment of nonbinding mucoid strains of R aeruginosa by testing various monosaccharides or lectins for inhibition of adherence; they dem:> e; SAGA 3606 onstrated that GaINAc, galactose, and binding (injured) 'b8 NANA werethe best inhibitors of adherx ence of mucoid strains of R aeruginosa, .~ and that lectins could interact with the ; ti 4 mucoid organisms and the host. They ~ _ ......_ .... binding (intact) '0 2 __ ......- - ._.J.1J nonbinding hypothesized that the mucoid exopoly... -~:::.-.-._.-.-.saccharides of mucoid strains of R aeru.lJ ginosawere adhesins that interacted with E ;:, z the glycocalyx on the airway surface. SAGA 3760 Ramphal and colleagues (16), Sato and 8 Okinaga (17), and Woods and coworkbinding (injured) ers (18), on the other hand, demonstrated 6 that the presence of pili was important 4 ~ binding (intact) for nonmucoid strains of R aeruginosa _ ... :::,"'::";;(i1 nonbinding to bind to the airway surface. Moreover, 2 :,":.:::,".:::::,":.::.-' Irvin and colleagues (19) and Doig and coworkers (20) recently demonstrated o 90 that monoclonal antibody against pili inIncubation Time (min) hibited attachment of R aeruginosa to Fig. 4. Growth of P. aeruginosa (ATCC 27853, SAGA cells, and that the pilin structural pro3606, and SAGA 3760) after binding to rat trachea. Tratein subunit has a cell-binding function cheal preparations were exposed to bacterial solution, which was adjusted to an optical density of 0.20 in the as the pilus adhesin. These data indicate intact trachea group or an optical density of 0.02 in the that the pili of nonmucoid strains of R brush-injured trachea group. After bacterial exposure, aeruginosa play an important role in the each tracheal preparation was incubated for 90 min at attachment process. Ramphal and Pyle 310 C in 1.0 ml Hanks' solution containing 10% FBS. The grow1hof nonbinding bacteria also was measured (21) pointed out that R aeruginosa inas control. Each point represents the mean of three fection occurred when the bronchial animals before and after incubation. Solid circles = mucosa was damaged. In animal experibinding bacteria to intact trachea; open circles = bindments, they have demonstrated that R ing bacteria to brush-injured trachea; dotted circles = aeruginosa binds to acid-injured trachea nonbinding bacteria. more often than does E. coli or Klebsiellapneumoniae (5). In addition, they carcreased from 1.0 X 104 to 2.5 X 104 ried out a partial characterization of the cfu/O.1 ml (ATCC 27853), from 0.9 X binding sites on acid-injured trachea for 104 to 2.0 X 104 cfu/O.1 ml (SAGA 3606), this organism (22). In clinical terms, howand from 0.9 X 104 to 3.1 X 104 cfu/O.1 ever, most of tracheal damages are meml (SAGA 3760). In each strain, bacte- chanical injuries such as caused by inturia binding to brush-injured trachea grew bation or suction tubes, especially when more rapidly than either those binding patients are kept under management with to intact trachea or nonbinding bacteria. intubation. Tothe best of our knowledge, there have been no reports dealing with Discussion the binding sites for R aeruginosa inA tropism of bacteria for a given organ duced by mechanical injury. In the present study, we first meahas recently been recognized as an important factor in the development of in- sured bacterial attachment of nonmucoid fections. In urinary tract infections, in strains of R aeruginosa to rat tracheal fact, it has been shown that Escherichia surface. Tracheal surface injury effected coli binds to urinary tract surface with by brushing significantly increased the various types of pili, and that the recep- number of bacteria binding to the surtor on tissue surface for this organism face (table 1). This enhancement of bacconsists of glycoconjugates (12-15). In terial adherence clearly shows that the the respiratory tract infections, on the mechanical injury on the tracheal surother hand, there has only been a small face increases the binding sites for R amount of evidence for this phenome- aeruginosa. Our histologic observations ATCC 27853
8
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1151
(figure IB) have demonstrated that most of the binding sites on the injured tracheal surface are on naked submucosal connective tissue. We also carried out a partial characterization of the binding sites of R aeruginosa on either intact or brush-injured rat tracheal surface. For that purpose, we used metaperiodate, which oxidizes carbohydrates, and trypsin, which destroys protein components. Treatment of tracheal surface with 90 mM metaperiodate significantly decreased the number of bacteria binding to injured trachea (19 to 260/0 of control) but trypsin treatment did not (figure 2B). In contrast, neither reagent had any effect on bacterial attachment to intact tracheal surface (figure 2A). These data show that the binding sites for R aeruginosa on brushinjured tracheal surfaces are different from those on intact trachea. We have concluded that brush injury on tracheal surfaces produces new binding sites for R aeruginosa as a receptor, which is not availablewith intact trachea, and that this receptor consists of carbohydrates that are important components and that it is not a protein receptor. In an attempt to determine whether the inhibiting sugars werebinding to the bacterial adhesins, bacteria were incubated with various sugars before bacterial attachment (table 2). Mannose, fucose, GaINAc, and G1cNAc did not have any effect on bacterial attachment, whereas NANA significantly inhibited bacterial adherence. However, the growth of bacteria that are treated with monosaccharides has demonstrated that NANA is an agent strongly toxic to R aeruginosa (figure 3). It is therefore suggested that the inhibition of bacterial attachment with NANA does not indicate specific blocking of binding between pili and a sugar of the receptor. The neuraminidase and lectin data also support this result (table 2). Treatment of injured tracheal surfaces with either neuraminidase type III or V, which cleavesNANA, or L. polyphemus lectin, which agglutinates NANA, had little effect on bacterial attachment. Thus, it is still unclear what kind of sugar is involved here as an important component of the receptor on brush-injured tracheal surface for R aeruginosa. At least, it has been confirmed that this receptor consists of carbohydrates. Let us now turn to bacterial growth. Bacteria must grow, after binding to tissue, to develop an infection. Accordingly, we took an in vitro measurement of bacterial growth during a 90-min incu-
1152
YAMAGUCHI AND YAMADA
bation period following bacterial binding to tracheal surface. It has been reported that many bacteria grow tenaciously once they have adhered to a solid surface. Henrici and Johnson (23), for instance, reported several species of bacteria that grewonly when attached to a firm surface, and Zobell (24) argues that attachment to a solid surface is the basis for bacterial growth. Wealso demonstrated, in a previous study (25), that P. aeruginosa binding to human tracheobronchial mucin coating on glass tubes grew more rapidly than their nonbinding counterparts. In this current study, bacteria binding to brush-injured trachea grew more rapidly than either nonbinding bacteria or those on intact trachea. This phenomenon is probably attributable to the mechanism pointed out by Zobell (24), who argues that bacterial growth after binding depends on certain surface conditions including electric charge, pH, and other factors. Our data suggest that mechanical damage, like brush injury on airway surface, induces a promoting effect on the attachment and growth of P. aeruginosa. We emphasize that airway suface damage plays an important role in the pathogenesis of lesions caused by P. aeruginosa with production of the receptors for this particular organism. References 1. Niederman MS. Ferranti RD. Zeigler A, Merrill WW, Reynolds HY. Respiratory infection complicating long-term tracheostomy. The implication of persistent gram-negative tracheobronchial
colonization. Chest 1984; 85:39-44. 2. Bryant LR, Trinkle JK, Mobin-Uddin K, Baker J. Griffen WOo Bacterialcolonization profile with tracheal intubation and mechanical ventilation. Arch Surg 1972; 104:647-51. 3. Brook I. Bacterial colonization, tracheobronchitis, and pneumonia followingtracheostomy and long-term intubation in pediatric patients. Chest 1979; 76:420-4. 4. Ramphal R. Small PM. Shands JW, Fischlschweiger W. Small PA. Adherence of Pseudomonasaeruginosa to tracheal cells injured by influenza infection or by endotracheal intubation. Infect Immun 1980; 27:614-9. 5. Ramphal R, Vishwanath S. Why is Pseudomonas the colonizer and why does it persist? Infection 1987; 15:281-7. 6. Johanson WG, Higuchi JH, Chaudhuri TR, Woods DE. Bacterial adherence to epithelial cells in bacillary colonization of the respiratory tract. Am Rev Respir Dis 1980; 121:55-63. 7. Franklin AL, Todd T, Gurman G, Black D, Mankinen-Irvin PM. Irvin RT. Adherence of Pseudomonas aeruginosa to cilia of human tracheal epithelial cells. Infect Immun 1987; 55:1523-5. 8. Niederman MS, RaffertyTD, Sasaki CT, Merrill WW. Matthay RA. Reynolds HY. Comparison of bacterial adherence to ciliated and squamous epithelial cells obtained from the human respiratory tract. Am Rev Respir Dis 1983; 127:85-90. 9. Ramphal R, Pyle M. Adherence of mucoid and nonmucoid Pseudomonas aeruginosa to acidinjured tracheal epithelium. Infect Immun 1983; 41:345-51. 10. Marcus H. Austria Al., Baker NR. Adherence of Pseudomonas aeruginosa to tracheal epithelium. Infect Immun 1989; 57: 1050-3. 11. Marcus H. Baker NR. Quantitation of adherence of mucoid and nonmucoid Pseudomonas aeruginosa to hamster tracheal epithelium. Infect Immun 1985; 47:723-9. 12. Ofek I, Mirelman D, Sharon, N. Adherence of Escherichia coli to human mucosal cells mediated by mannose receptors.Nature 19077; 265:623-5. 13. Virkola R. Westerlund B, Holthafer H, Parkkinen J. KekomakiM, Korhonen TK. Binding characteristics of Escherichia coli adhesins in human
urinary bladder. Infect Immun 1988; 56:2615-22. 14. Gander RM. Thomas VL. Distribution of type 1 and P pili on uropathogenic Escherichia coli06. Infect Immun 1987; 55:293-7. 15. Korhonen TK. Vaisanen-Rhen V. Rhen M. Pere A, Parkkinen J, Finne J. Escherichia coli fimbriae recognizing sialyl galactosides. J Bacteriol 1984; 159: 762-6. 16. Ramphal R. Sadoff JC. Pyle M. Silipigni JD. Role of pili in the adherence of Pseudomonasaeruginosa to injured tracheal epithelium. Infect Immun 1984; 44:38-40. 17. Sato H, Okinaga K. Role of pili in the adherence of Pseudomonasaeruginosa to mouse epidermal cells. Infect Immun 1987; 55:1774-8. 18. Woods DE, Straus DC, Johanson WG, Berry VK. Bass JA. Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells. Infect Immun 1980; 29:1146-51. 19. Irvin RT,Doig P. Lee KK, et al. Characterization of the Pseudomonas aeruginosa pilus adhesin: confirmation that the pilin structural protein subunit contains a human epithelial cell-binding domain. Infect Immun 1989; 57:3720-6. 20. Doig P, Sastry PAt Hodges RS, LeeKK. Paranchych W. Irvin RT. Inhibition of pilus-mediated adhesion of Pseudomonas aeruginosa to human buccal epithelial cells by monoclonal antibodies directed against pili. Infect Immun 1990;58:124-30. 21. Ramphal R. Pyle M. Evidence for mucins and sialic acid as receptors for Pseudomonas aeruginosa in the lower respiratory tract. Infect Immun 1983; 41:339-44. 22. Vishwanath S, Ramphal R. Tracheobronchial mucin receptor for Pseudomonas aeruginosa: predominance of amino sugars in binding sites. Infect Immun 1985; 48:331-5. 23. Henrici AT,Johnson DE. Studies of freshwater bacteria. II. Stalked bacteria, a new order of schizomycetes. J Bacteriol 1935; 30:61-93. 24. Zobell CEo The effect of solid surfaces upon bacterial activity. J Bacteriol 1943; 45:39-56. 25. Katoh 0, Yamada H, Yamaguchi T, et al. Adherence of gram negative bacteria to human tracheobronchial mucin. Jpn J Thorac Dis 1988; 26:1182-6.
TSUNEKO YAMAGUCHI and HOZUMI YAMADA Introduction
In general, gram-negative bacteria are frequently cultured from patients with structural epithelial damage, such as results from prolonged endotracheal intubation, acid aspiration, and tracheostomy (1-4). In these colonizing bacteria, Pseudomonas aeruginosa is clinicallythe most important colonizer in the respiratory tract for its low susceptibility to antibiotics. Why does R aeruginosa colonize in the respiratory tract? In an attempt to answer this question, Ramphal and colleagues (5) suggested the following for consideration: (1) R aeruginosa has a tropism for the respiratory tract; (2) there must be a defect in physical clearance; and (3) there must be a defect in immune clearance. These three mechanisms must playa role in the colonization of this organism on airway surface. Although R aeruginosa has a tropism toward airway surface (6-8), it has been clinically recognized that colonization or infection by this organism occurs when bronchial mucosa is injured (4). And, in vitro, it has been revealedthat acid-injury on airway surface enhanced attachment of this organism (9). In clinical terms, however, most tracheal damages are mechanical injuries, such as caused by intubation or suction tubes, especially when patients are kept under management with intubation. To the best of our knowledge, however, there have been no reports dealing with the binding sites for R aeruginosa induced by mechanical injury on airway surface. In this report, we shall demonstrate an enhanced attachment of R aeruginosa by airway surface damage and an enhanced growth of this organism after binding to injured sites using an in vitro experiment system of rat trachea. Weshall also carry out a partial characterization of the receptor on brush-injured tracheal surface for this organism. Methods Bacterial Preparation Three strains of piliated, nonmucoid type of R aeruginosa (ATCC27853,SAGA 3606, and
SUMMARY In this study, bacterial attachment to rat tracheal surface was measured using three nonmucold strains of Pssudomonas asruglnosa and bacterial growth after binding to tracheal surface was also teste\j. Brush Injury on tracheal surface significantly Increased bacterial attachment (1,190to 1,600%); bacteria binding to brush-Injured sites grew more rapidly than either nonbinding bacteria or those on Intact trachea. A partial characterization of the binding sites for P. asruglnosa on either Intact or injured tracheal surface also was performed. Treatment of Injured tracheal surface with metaperlodate significantly Inhibited attachment of P. asruginosa, but trypsin treatment did not. In contrast, neither reagent had any effects on bacterial attachment to Intact tracheal surface. These resuIts suggest that brush Injury on tracheal surface produces new binding sites as a receptor for P. asruginosa, and that this receptor has carbohydrates as Important components and that It Is not a protein receptor. In addition, In order to determine what the dominant sugar of this receptor was, we tested the Inhibition of bacterial attachment with monosaccharide, neuraminldase, and lectin. Treatment of bacteria with N·acetylneuramlnlc acid (NANA) dramatically In· hibited bacterial attachment to injured trachea. However, NANA also inhibited the growth of this organism. Moreover, neither neuraminidase nor lectin data suggested that the dominant sugar of the receptor was NANA. Our data go so far as to confirm that the major component of the receptor of nonmucoid strains of P. asruglnosa on brush·injured trachea Is carbohydrates; It Is stili unclear what kind of sugar Is the dominant component of the receptor. We have concluded that mechanical injury, such as caused by brushing, Induces a promoting effect on attachment and growth of P. aeruginosa, and that this effect is attributed to production of the receptor that Is not available with Intact trachea. Such a tropism of P. aeruglnosa to airway surface Induced by mechanical injury Is likely to play an Important role in the colonization and Infection of this organism. AM REV RESPIR DIS 1991; 144:1147-1152
SAGA 3760) were used in this study. SAGA 3606 and SAGA 3760 strains were isolated from sputum. Each organism was grown in Trypticase soy broth (Becton Corp., Cockeysville, MD) for 16 h at 37° C, washed three times in sterile saline, resuspended in sterile saline, and then adjusted to an optical density of 0.07 for each strain at 650 nm in a spectrophotometer. These concentrations of bacterial solution contained approximately 3 x 107 colony-forming units (cfu)/ml.
Rat Tracheal Preparation A total of 192male Donryu rats, 11 to 14 wk old, were obtained from Kyudoh Corp. (Fukuoka, Japan). Animals were killed by intraperitoneal injection of 500 to 800 mg/kg pentobarbital, and then the trachea was excised and cut to 1.5 to 2.0 em in length. In the injured trachea group, tracheal surface was brushed with a small bronchoscopic brush (BC-IO; Olympus Corp., Tokyo, Japan). After brushing, the tracheal lumen was washed with 2 ml sterile saline. We then observed the turbidity caused by desquamative cells and tissue debris in each solution washed. Samples having the same degree of turbidity demonstrated a similar severity of histologic injury. Microscopically, specimens of brushinjured rat trachea presented severe damages
of epithelial cells; most ciliated cells were lost and submucosal tissues were naked.
Bacterial Attachment 'Testing In this study, we modified the adherence assay reported by Marcus and coworkers (10, 11), who carried out a quantitative analysis of bacterial adherence using a culture method. In the bacterial attachment testing, three non mucoid strains of R aeruginosa (ATCC 27853, SAGA 3606, and SAGA 3760) were used and five animals were killed in each group. Excised rat trachea was connected to small tubes 1.5mm in diameter. Tracheal surface was then rinsed with sterile saline, and a bacterial solution adjusted to the concentration of 3 x 107 cfu/ml was instilled into the tracheal lumen. Then the tracheal preparation wasincubated for 1hat 37° C in Hanks'
(Received in original form May 30, 1990 and in revised form May 1, 1991) I From the Department of Internal Medicine, Saga Medical School, Saga, Japan. 2 Correspondence and requests for reprints should be addressed to Dr. Hozumi Yamada, Department of Internal Medicine, Saga Medical School, I-I, Nabeshima 5-chome, Saga 849, Japan.
1147
1148
solution. In each experiment, a small volume of Hanks' solution, in which the tracheal preparation had been incubated for 1 h, was cultured to confirm no leakage of bacterial solution from the tracheal lumen. After a 1-h incubation, the tracheal lumen was rinsed with 120 ml sterile saline to wash out nonbinding bacteria. Byscanning electron microscopy,we ascertained that bacteria directly attached to intratracheal surface. For a quantitative analysis of binding bacteria, the tracheal preparation was homogenized in 2 ml of sterile saline. After huge materials were removed by meshes, a volume of 50 III of each homogenized solution was spread on a modified drigalski agar plate (BTB;Eiken Corp., Tokyo, Japan) using the Spiral System (Spiral System Inc., Cincinnati, OH). After overnight incubation, bacterial colonies on plates were counted with a Laser Bacteria Colony Counter (Spiral System). A quantitative analysis using a combination of both types of equipment presented reproducible data. Bacteria binding to tracheal surface were represented as a number of bacteria per square millimeter of tracheal surface.
Partial Characterization of Binding Sites The following reagents were obtained from Sigma Chemical Corp. (St. Louis, MO): trypsin, sodium metaperiodate, D - ( +)- mannose (mannose), L- (- ) - fucose (fucose), N-acetylneuraminic acid (NANA), N-acetylgalactosamine (GaINAc), N-acetylglucosamine (GIcNAc), Vibrio cholerae neuraminidase type III, Clostridium perfringens neuraminidase type V,Arachis hypogaealectin, Pisum sativum lectin type III, and Limuluspolyphemus lectin. In the characterization experiments, we used three nonmucoid strains of R aeruginosa (ATCC27853, SAGA 3606,and SAGA 3760)and killed three animals in each group. In order to perform a partial characterization of the binding sites for R aeruginosa on either intact or brush-injured tracheal surface, we used metaperiodate, which oxidizes carbohydrates, and trypsin, which destroys protein components. Rat tracheal surface was rinsed with 20 ml sterilesaline and then treated with 90 mM sodium metaperiodate (pH 5.0) for 40 min at room temperature or with 20 ug/ml trypsin for 15min at 25 0 C. In the control group, we used sterile saline instead of either reagent. After treatment with a reagent, the tracheal surface was rinsed with 60 ml sterile saline to wash out the reagent completely. The control tracheal preparations also were rinsed with sterile saline before exposure to bacteria. Bacterial attachment testing was then carried out as described previously. The results were represented as a number of bacteria per square millimeter 0 f tracheal surface. In addition, weused monosaccharides, neuraminidases, and lectins to perform a further characterization of the binding sites for R aeruginosa on brush-injured tracheal surface. In the monosaccharide experiments, R aeruginosa was incubated for 1 h at 370 C with
YAMAGUCHI AND YAMADA
various sugars: mannose (100 mM), fucose (100 mM), GIcNAc (100 mM), GalNAc (100 mM), and NANA (1 mM). In the control group, bacteria were incubated in sterile saline for 1 hat 37 0 C. After incubation, bacteria were washed three times in sterile saline and then bacterial attachment testing was carried out with injured tracheal preparations. The result of bacterial attachment was represented as a ratio(%) to control. Moreover, the growth of R aeruginosa treated with each monosaccharide was measured as the optical density changed while organisms were incubated in Hanks' solution containing 10% fetal bovine serum(FBS) at 370 C. In the neuraminidase and lectin experiments, injured tracheal surface was treated with 0.2 U neuraminidase type III in 0.15M NaCI and 4 mM CaCl 2 (pH 5.5) or 2.0 U neuraminidase type V in 50 mM sodium acetate (pH 4.9) for 40 min at 37 0 C in Hanks' solution; it also was treated with 100 ug/ml of A. hypogaea lectin, R sativum lectin type III, and L. polyphemus lectin for 40 min at 25 0 C. In both experiments, we used sterile saline instead of a reagent in the control group. After treatment with either reagent, the tracheal surface was rinsed with 60 ml sterile saline to wash out the reagent. The control tracheal preparations also were rinsed with sterile saline before bacterial exposure. Bacterial attachment testing was then carried out. The result of bacterial attachment was represented as a ratio (%) to control.
Bacterial Growth Testing For the growth testing of bacteria binding to rat tracheal surface, three strains of nonmucoid R aeruginosa (ATCC27853, SAGA 3606, and SAGA 3760)wereused and three animals were killed in each group. The tracheal preparation was exposed to bacterial solution, which was adjusted to an optical density of 0.20 in the intact trachea group or to an optical density of 0.02 in the brush-injured trachea group. After a 1-hexposure, the tracheal lumen was rinsed with sterile saline, and then the tracheal preparation was cut into two pieces; one piece was homogenized immediately for the counting of binding bacteria and the other was incubated for 90 min at 37 0 C in 1 ml Hanks' solution containing 10% FBS to observe bacterial growth after binding. After the 90-min incubation, the tracheal preparation was homogenized. The number of bacteria was counted with the same management as described above.The results wererepresented as a number of bacteria per mIlt of tracheal surface. On the other hand, the growth of nonbinding bacteria also was measured as control when a total number of approximately 1.0 x 104 cfu/O.l ml of each strain was incubated for 1 h at 37 0 C in 1 ml Hanks' solution including 10% FBS. The results were represented as numbers of bacteria per 0.1 ml. Statistical Analysis Each result represented the mean ± standard deviation (SD) of five animals for bacterial
attachment; the mean of three animals was used for the characterization of binding sites. Statistical analysis was performed by the unpaired Student's t test.
Results
Bacterial Attachment to Tracheal Surface Bacterial attachment to tracheal surface was tested using three strains of nonmucoid R aeruginosa. The results of this testing are shown in table 1. The numbers of bacteria binding to intact trachea were 4.4 ± 1.4 x 103 cfu/mm? for strain ATCC 27853, 4.4 ± 1.2 x 103 cfu/mmfor SAGA 3606, and 3.9 ± 1.7 x 103 cfu/mm? for SAGA 3760. The number of bacteria binding to tracheal surface significantly increased by brush-injury (ATCC 27853 = 1,190070 of intact, p < 0.01; SAGA 3606 = 1,290070 of intact, p < 0.01; SAGA 3760 = 1,600070 of intact, p < 0.01). Figure 1 shows the electron microscopic findings of bacterial attachment on intact tracheal surface (figure lA) and on brush-injured tracheal surface (figure IB). A few bacteria are found on cilia of intact epithelial cells (figure lA). In contrast, figure IB shows that most of the epithelial cells are lost and a huge number of bacteria are binding to naked submucosal connective tissue directly. Characterization of Binding Sites In order to perform a partial characterization of the binding sites on either intact or brush-injured tracheal surface, we tested the influence on bacterial attachment of metaperiodate, which oxidizes carbohydrates and trypsin, which destroys protein components. Effects of both reagents on bacterial attachment are shown in figure 2. Treatment with 90 mM metaperiodate significantly reduced the TABLE 1 EFFECTS OF BRUSH INJURY OF TRACHEAL SURFACE ON BACTERIAL ATIACHMENT TO RAT TRACHEA·
Nonmucoid Strains of P. aeruginosa ATCC 27853 SAGA 3606 SAGA 3760
No. of Bacteria Binding to Trachea (x 103 cfu/mm2) Intact
Brush-injured
4.4 ± 1.4 4.4 ± 1.2 3.9 ± 1.7
52.4 ± 11.2t 56.8 ± 15.9t 62.7 ± 11.8t
• Three nonmucoid strains of P. aeruginosa were tested and tracheal surface was injured with a bronchoscopic brush. Binding bacteria are represented as a number of bacteria per square millimeter of tracheal surface. Each result represents the mean ± SO of five animals. Statistical analysis was performed by the unpaired Student's t test.
t p < 0.01.
AIRWAY SURFACE DAMAGE AND PATHOGENESIS OF P. AERUGINOSA
1149
Fig. 1. Intact tracheal surface is covered with normal ciliated epithelial cells and a small number of P. aeruginosa (ATCC27853) are found on cilia (A). In contrast, brush-injured tracheal surface has completely lost ciliated epithelial cells and submucosal connective tissue is naked. Huge numbers of bacteria (ATCC 27853) have directly attached to submucosal connective tissue (B).
(AI
number of bacteria binding to brushinjured trachea (ATCC 27853, 19% of control; SAGA 3606, 26070 of control; SAGA 3760, 22% of control), but trypsin treatment had little effect on bacterial attachment (ATCC 27853, 109% of control; SAGA 3606, 92010 of control; SAGA 3760, 74% of control). In contrast, neither reagent had any effect on bacterial attachment to intact tracheal surface (metaperiodate: ATCC 27853, 110070 of control; SAGA 3606, 134% of control; SAGA 3760, 159% of control; trypsin: ATCC 27853, 86% of control; SAGA 3606, 77% of control; SAGA 3760, 141 % of control). These data reveal that the binding sites for R aeruginosa on injured tracheal surface are different from those on intact tracheal surface. It is concluded that brush injury on tracheal surface produces newbinding sites for R aeruginosa as a receptor, and that this receptor consists of carbo-
ATCC27853
5AGA3606
5AGA3760
1.0
0.8
Fig. 2. Effects of metaperiodate or trypsin on attachment of three nonmucoid strains of P. aeruginosa to either intact (A) or brush-injured (B ) trachea. Before bacterial exposure, rat trachea was treated with 90 mM metaperiodate (solid bars) or 20 lJ.9/ml of trypsin (hatched bars). In the control group, tracheal preparations were also treated with sterile saline (open bars) instead of either reagent. Bacteria (3 x 107 cfu/ml) were exposed to rat tracheal surface for 1 h at yo C. Binding bacteria (ordinate) represent the mean ± SD ofthree animals. Statistical analysis was performed by the unpaired Student's t test. Asterisks indicate significant values of difference compared with control (p < 0.01).
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1150
hydrates that are an important component and that it is not a protein receptor. For a further characterization of the receptors produced by brushing, we also tested the influence on bacterial attachment of monosaccharide, neuraminidase, and lectin (table 2). Mannose (100mM), fucose (100mM), GlcNAc (100mM), and GalNAc (100mM) had little effect on attachment of these nonmucoid strains of R aeruginosa to injured tracheal surface, but 1mM NANA significantly inhibited their attachment (ATCC 27853, 5070 of control; SAGA 3606, 10070 of control; SAGA 3760,3% of control). On the other hand, figure 3 shows the growth of R aeruginosa after treatment with each of the monosaccharides tested. Mannose, fucose, GlcNAc, and GalNAc had hardly any effect on the growth of R aeruginosa, but NANA dramatically inhibited the growth of this organism. The effects of two different types of neuraminidase on bacterial attachment are given in table 2. JI: cholerae neuraminidase type III (0.2 U) did not decrease bacterial attachment to injured trachea (ATCC 27853, 88% of control; SAGA 3606, 105% of control; SAGA 3760, 104% of control), nor did C perfringens neuraminidase type V (2 U) decrease bacterial attachment (ATCC 27853, 73070 of control; SAGA 3606, 115070 of control; SAGA 3760, 106% of control). The lectin data are also shown in table 2. Treatment of injured tracheal surface with lectins from A. hypogaea, (103 to 151070 of control), R sativum, (80 to 109% of control), and L. polyphemus (85 to 107% of control), did not inhibit attachment of these organisms.
Bacterial Growth Testing R aeruginosa binding to rat trachea was incubated for 90 min in Hanks' solution containing 10070 FBS. The numbers of binding bacteria before and after the 90min incubation are shown in figure 4. During the 90-min incubation, bacteria on intact trachea increased from 1.6 X 104 to 3.1 X 104 cfu/mm" (ATCC 27853), from 1.2 X 104 to 3.6 X 104 cfu/mm" (SAGA 3606), and from 1.2 X 104 to 3.1 X 104 cfu/mm? (SAGA 3760); those on brush-injured trachea increased from 0.8 X 104 to 4.2 X 104 cfu/mm-, from 1.2 x 104 to 8.6 X 104 cfu/mm-, and from 1.0 X 104 to 6.4 X 104 cfu/mm-, respectively. On the other hand, the growth of nonbinding bacteria also was measured as control in each strain and the result is also shown in figure 4. During the 90min incubation, nonbinding bacteria in-
YAMAGUCHI AND YAMADA
TABLE 2 EFFECTS OF MONOSACCHARIDE, NEURAMINIDASE, AND LECTIN ON ATIACHMENT OF P. AERUGINOSA TO BRUSH-INJURED TRACHEA' Attachment of P. aeruginosa to Brush-injured Trachea (%)
Reagents Monosaccharide Mannose, 100 mM Fucose, 100 mM GlcNAc, 100 mM GaINAc, 100 mM NANA, 1 mM Neuraminidase V. cho/erae, 0.2 U C. perfringens, 2 U Lectin A. hypogaea, 100 ~g/ml P. sa tivum , 100 ~g/ml L. polyphemus, 100 ~g/ml
ATCC 27853
SAGA 3606
SAGA 3760
98 115 76 105 5
86 124 85 92 10
114 90 101 97 3
88 73
105 115
104 106
151 109 107
132 80 85
103 93 89
Definition of abbreviations: GlcNAc = N-acetylglucosamine; GalNAC = N-acetylgalactosamine; NANA = N-acetylneuraminic acid; V. cholerae = Vibrio cholerae type III; C. perfringens = Clostridium perfringens type V; A. hypogaea = Arachis hypogaea; P. sativum = Pisum sativum type III; L. polyphemus = Limulus polyphemus. • In the monosaccharide testing, bacteria were incubated with each monosaccharide before exposure to injured trachea. In the neuraminidase and lectin testing, injured tracheal preparations were treated with neuraminidase or lectin before bacterial exposure. The results of bacterial attachment were represented as a ratio (%) to control using saline instead of a reagent. Each result represents the mean of three animals.
ATCC 27853
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Fig. 3. Growth of P. aeruginosa (ATCC 27853, SAGA 3606, and SAGA 3760) after treatment with various monosaccharides: mannose (100 mM), fucose (100 mM), GlcNAc (100 mM), GalNAc (100 mM), and NANA (1 mM). Control bacteria were treated with sterile saline instead of monosaccharides. Bacterial growth was measured according to how the optical density changed in Hanks' solution cdntaining 10% FBS while incubated at 37" C.
AIRWAY SURFACE DAMAGE AND PATHOGENESIS OF P. AERUGINOSA
non. Previous investigators reported that a tropism of the respiratory tract on the part of R aeruginosa plays an important 6 role in the development of infections binding (injured) 4 (6-8). Marcus and colleagues (10)exam_ ... binding (intact) ined the mechanism of attachment of nonbinding mucoid strains of R aeruginosa by testing various monosaccharides or lectins for inhibition of adherence; they dem:> e; SAGA 3606 onstrated that GaINAc, galactose, and binding (injured) 'b8 NANA werethe best inhibitors of adherx ence of mucoid strains of R aeruginosa, .~ and that lectins could interact with the ; ti 4 mucoid organisms and the host. They ~ _ ......_ .... binding (intact) '0 2 __ ......- - ._.J.1J nonbinding hypothesized that the mucoid exopoly... -~:::.-.-._.-.-.saccharides of mucoid strains of R aeru.lJ ginosawere adhesins that interacted with E ;:, z the glycocalyx on the airway surface. SAGA 3760 Ramphal and colleagues (16), Sato and 8 Okinaga (17), and Woods and coworkbinding (injured) ers (18), on the other hand, demonstrated 6 that the presence of pili was important 4 ~ binding (intact) for nonmucoid strains of R aeruginosa _ ... :::,"'::";;(i1 nonbinding to bind to the airway surface. Moreover, 2 :,":.:::,".:::::,":.::.-' Irvin and colleagues (19) and Doig and coworkers (20) recently demonstrated o 90 that monoclonal antibody against pili inIncubation Time (min) hibited attachment of R aeruginosa to Fig. 4. Growth of P. aeruginosa (ATCC 27853, SAGA cells, and that the pilin structural pro3606, and SAGA 3760) after binding to rat trachea. Tratein subunit has a cell-binding function cheal preparations were exposed to bacterial solution, which was adjusted to an optical density of 0.20 in the as the pilus adhesin. These data indicate intact trachea group or an optical density of 0.02 in the that the pili of nonmucoid strains of R brush-injured trachea group. After bacterial exposure, aeruginosa play an important role in the each tracheal preparation was incubated for 90 min at attachment process. Ramphal and Pyle 310 C in 1.0 ml Hanks' solution containing 10% FBS. The grow1hof nonbinding bacteria also was measured (21) pointed out that R aeruginosa inas control. Each point represents the mean of three fection occurred when the bronchial animals before and after incubation. Solid circles = mucosa was damaged. In animal experibinding bacteria to intact trachea; open circles = bindments, they have demonstrated that R ing bacteria to brush-injured trachea; dotted circles = aeruginosa binds to acid-injured trachea nonbinding bacteria. more often than does E. coli or Klebsiellapneumoniae (5). In addition, they carcreased from 1.0 X 104 to 2.5 X 104 ried out a partial characterization of the cfu/O.1 ml (ATCC 27853), from 0.9 X binding sites on acid-injured trachea for 104 to 2.0 X 104 cfu/O.1 ml (SAGA 3606), this organism (22). In clinical terms, howand from 0.9 X 104 to 3.1 X 104 cfu/O.1 ever, most of tracheal damages are meml (SAGA 3760). In each strain, bacte- chanical injuries such as caused by inturia binding to brush-injured trachea grew bation or suction tubes, especially when more rapidly than either those binding patients are kept under management with to intact trachea or nonbinding bacteria. intubation. Tothe best of our knowledge, there have been no reports dealing with Discussion the binding sites for R aeruginosa inA tropism of bacteria for a given organ duced by mechanical injury. In the present study, we first meahas recently been recognized as an important factor in the development of in- sured bacterial attachment of nonmucoid fections. In urinary tract infections, in strains of R aeruginosa to rat tracheal fact, it has been shown that Escherichia surface. Tracheal surface injury effected coli binds to urinary tract surface with by brushing significantly increased the various types of pili, and that the recep- number of bacteria binding to the surtor on tissue surface for this organism face (table 1). This enhancement of bacconsists of glycoconjugates (12-15). In terial adherence clearly shows that the the respiratory tract infections, on the mechanical injury on the tracheal surother hand, there has only been a small face increases the binding sites for R amount of evidence for this phenome- aeruginosa. Our histologic observations ATCC 27853
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QI
1151
(figure IB) have demonstrated that most of the binding sites on the injured tracheal surface are on naked submucosal connective tissue. We also carried out a partial characterization of the binding sites of R aeruginosa on either intact or brush-injured rat tracheal surface. For that purpose, we used metaperiodate, which oxidizes carbohydrates, and trypsin, which destroys protein components. Treatment of tracheal surface with 90 mM metaperiodate significantly decreased the number of bacteria binding to injured trachea (19 to 260/0 of control) but trypsin treatment did not (figure 2B). In contrast, neither reagent had any effect on bacterial attachment to intact tracheal surface (figure 2A). These data show that the binding sites for R aeruginosa on brushinjured tracheal surfaces are different from those on intact trachea. We have concluded that brush injury on tracheal surfaces produces new binding sites for R aeruginosa as a receptor, which is not availablewith intact trachea, and that this receptor consists of carbohydrates that are important components and that it is not a protein receptor. In an attempt to determine whether the inhibiting sugars werebinding to the bacterial adhesins, bacteria were incubated with various sugars before bacterial attachment (table 2). Mannose, fucose, GaINAc, and G1cNAc did not have any effect on bacterial attachment, whereas NANA significantly inhibited bacterial adherence. However, the growth of bacteria that are treated with monosaccharides has demonstrated that NANA is an agent strongly toxic to R aeruginosa (figure 3). It is therefore suggested that the inhibition of bacterial attachment with NANA does not indicate specific blocking of binding between pili and a sugar of the receptor. The neuraminidase and lectin data also support this result (table 2). Treatment of injured tracheal surfaces with either neuraminidase type III or V, which cleavesNANA, or L. polyphemus lectin, which agglutinates NANA, had little effect on bacterial attachment. Thus, it is still unclear what kind of sugar is involved here as an important component of the receptor on brush-injured tracheal surface for R aeruginosa. At least, it has been confirmed that this receptor consists of carbohydrates. Let us now turn to bacterial growth. Bacteria must grow, after binding to tissue, to develop an infection. Accordingly, we took an in vitro measurement of bacterial growth during a 90-min incu-
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bation period following bacterial binding to tracheal surface. It has been reported that many bacteria grow tenaciously once they have adhered to a solid surface. Henrici and Johnson (23), for instance, reported several species of bacteria that grewonly when attached to a firm surface, and Zobell (24) argues that attachment to a solid surface is the basis for bacterial growth. Wealso demonstrated, in a previous study (25), that P. aeruginosa binding to human tracheobronchial mucin coating on glass tubes grew more rapidly than their nonbinding counterparts. In this current study, bacteria binding to brush-injured trachea grew more rapidly than either nonbinding bacteria or those on intact trachea. This phenomenon is probably attributable to the mechanism pointed out by Zobell (24), who argues that bacterial growth after binding depends on certain surface conditions including electric charge, pH, and other factors. Our data suggest that mechanical damage, like brush injury on airway surface, induces a promoting effect on the attachment and growth of P. aeruginosa. We emphasize that airway suface damage plays an important role in the pathogenesis of lesions caused by P. aeruginosa with production of the receptors for this particular organism. References 1. Niederman MS. Ferranti RD. Zeigler A, Merrill WW, Reynolds HY. Respiratory infection complicating long-term tracheostomy. The implication of persistent gram-negative tracheobronchial
colonization. Chest 1984; 85:39-44. 2. Bryant LR, Trinkle JK, Mobin-Uddin K, Baker J. Griffen WOo Bacterialcolonization profile with tracheal intubation and mechanical ventilation. Arch Surg 1972; 104:647-51. 3. Brook I. Bacterial colonization, tracheobronchitis, and pneumonia followingtracheostomy and long-term intubation in pediatric patients. Chest 1979; 76:420-4. 4. Ramphal R. Small PM. Shands JW, Fischlschweiger W. Small PA. Adherence of Pseudomonasaeruginosa to tracheal cells injured by influenza infection or by endotracheal intubation. Infect Immun 1980; 27:614-9. 5. Ramphal R, Vishwanath S. Why is Pseudomonas the colonizer and why does it persist? Infection 1987; 15:281-7. 6. Johanson WG, Higuchi JH, Chaudhuri TR, Woods DE. Bacterial adherence to epithelial cells in bacillary colonization of the respiratory tract. Am Rev Respir Dis 1980; 121:55-63. 7. Franklin AL, Todd T, Gurman G, Black D, Mankinen-Irvin PM. Irvin RT. Adherence of Pseudomonas aeruginosa to cilia of human tracheal epithelial cells. Infect Immun 1987; 55:1523-5. 8. Niederman MS, RaffertyTD, Sasaki CT, Merrill WW. Matthay RA. Reynolds HY. Comparison of bacterial adherence to ciliated and squamous epithelial cells obtained from the human respiratory tract. Am Rev Respir Dis 1983; 127:85-90. 9. Ramphal R, Pyle M. Adherence of mucoid and nonmucoid Pseudomonas aeruginosa to acidinjured tracheal epithelium. Infect Immun 1983; 41:345-51. 10. Marcus H. Austria Al., Baker NR. Adherence of Pseudomonas aeruginosa to tracheal epithelium. Infect Immun 1989; 57: 1050-3. 11. Marcus H. Baker NR. Quantitation of adherence of mucoid and nonmucoid Pseudomonas aeruginosa to hamster tracheal epithelium. Infect Immun 1985; 47:723-9. 12. Ofek I, Mirelman D, Sharon, N. Adherence of Escherichia coli to human mucosal cells mediated by mannose receptors.Nature 19077; 265:623-5. 13. Virkola R. Westerlund B, Holthafer H, Parkkinen J. KekomakiM, Korhonen TK. Binding characteristics of Escherichia coli adhesins in human
urinary bladder. Infect Immun 1988; 56:2615-22. 14. Gander RM. Thomas VL. Distribution of type 1 and P pili on uropathogenic Escherichia coli06. Infect Immun 1987; 55:293-7. 15. Korhonen TK. Vaisanen-Rhen V. Rhen M. Pere A, Parkkinen J, Finne J. Escherichia coli fimbriae recognizing sialyl galactosides. J Bacteriol 1984; 159: 762-6. 16. Ramphal R. Sadoff JC. Pyle M. Silipigni JD. Role of pili in the adherence of Pseudomonasaeruginosa to injured tracheal epithelium. Infect Immun 1984; 44:38-40. 17. Sato H, Okinaga K. Role of pili in the adherence of Pseudomonasaeruginosa to mouse epidermal cells. Infect Immun 1987; 55:1774-8. 18. Woods DE, Straus DC, Johanson WG, Berry VK. Bass JA. Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells. Infect Immun 1980; 29:1146-51. 19. Irvin RT,Doig P. Lee KK, et al. Characterization of the Pseudomonas aeruginosa pilus adhesin: confirmation that the pilin structural protein subunit contains a human epithelial cell-binding domain. Infect Immun 1989; 57:3720-6. 20. Doig P, Sastry PAt Hodges RS, LeeKK. Paranchych W. Irvin RT. Inhibition of pilus-mediated adhesion of Pseudomonas aeruginosa to human buccal epithelial cells by monoclonal antibodies directed against pili. Infect Immun 1990;58:124-30. 21. Ramphal R. Pyle M. Evidence for mucins and sialic acid as receptors for Pseudomonas aeruginosa in the lower respiratory tract. Infect Immun 1983; 41:339-44. 22. Vishwanath S, Ramphal R. Tracheobronchial mucin receptor for Pseudomonas aeruginosa: predominance of amino sugars in binding sites. Infect Immun 1985; 48:331-5. 23. Henrici AT,Johnson DE. Studies of freshwater bacteria. II. Stalked bacteria, a new order of schizomycetes. J Bacteriol 1935; 30:61-93. 24. Zobell CEo The effect of solid surfaces upon bacterial activity. J Bacteriol 1943; 45:39-56. 25. Katoh 0, Yamada H, Yamaguchi T, et al. Adherence of gram negative bacteria to human tracheobronchial mucin. Jpn J Thorac Dis 1988; 26:1182-6.
