Pseudomonas aeruginosa variants isolated from patients with cystic fibrosis are killed by a bactericidal protein from human polymorphonuclear leukocytes.

The susceptibility of paired mucoid and nonmucoid variants of Pseudomonas aeruginosa isolated from 13 patients with cystic fibrosis (CF) to killing by a 55,000-Da bactericidal protein (BP55) from human polymorphonuclear leukocytes was studied. Mucoid and nonmucoid variants were equally sensitive to killing by BP55 at both pH 5.6 and pH 7.2. Eleven of the isolates were resistant to the bactericidal activity of 10% normal human serum but were as sensitive as the serum-sensitive isolates to BP55. Similarly, the 15 isolates with lipopolysaccharides (LPS) containing O-polysaccharide side chains (smooth LPS) were as sensitive to BP55 as those isolates with rough LPS.P. aeruginosa isolates from patients in poor clinical condition were more likely to have LPS of the smooth type and to be resistant to killing by 10% human serum than the isolates from patients in good clinical condition. We have concluded that the susceptibility of the P. aeruginosa isolates from patients with CF to killing by BP55 does not correlate with mucoid or nonmucoid variations, with the presence or absence of smooth LPS, or with the sensitivity or resistance to killing by normal human serum.     

Mucoid Pseudomonas aeruginosa in cystic fibrosis: characterization of muc mutations in clinical isolates and analysis of clearance in a mouse model of respiratory infection.

A distinguishing feature of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients is their mucoid, exopolysaccharide alginate-overproducing phenotype. One mechanism of conversion to mucoidy is based on mutations in the algU mucABCD cluster, encoding the stress sigma factor AlgU and its regulators. However, conversion to mucoidy in laboratory strains can be achieved via mutations in other chromosomal sites. Here, we investigated mechanisms of the emergence of mucoid P. aeruginosa in CF by analyzing the status of mucA in a collection of mucoid P. aeruginosa isolates from 53 CF patients. This negative regulator of algU, when inactivated under laboratory conditions, causes conversion to mucoidy. The overall frequency of mucA alterations in mucoid CF isolates was 84%. Nucleotide sequence analyses revealed that the majority of the alterations caused premature termination of the mucA coding sequence. Comparison of paired nonmucoid and mucoid P. aeruginosa isolates from three CF patients indicated the presence of mucA mutations only in the mucoid strains. Interestingly, mucoid P. aeruginosa isolates from urinary tract infections also had mutations in the mucA gene. Clearance of CF isolates from the murine lung was investigated in an aerosol infection model with C57BL/6J, BALB/c, and DBA/2NHsd mice. Two CF strains, selected for further study based on the dependence of their alginate production on the concentration of salt in the medium, were used to examine the effects of mucoidy on pulmonary clearance. Statistically significant improvement in recovery from the murine lung of viable mucoid P. aeruginosa cells relative to the nonmucoid bacteria was observed in the majority of mouse strains tested. Collectively, the results reported here suggest that mucA is most likely the preferential site for conversion to mucoidy in CF and that alginate overproduction in mucA-mutant P. aeruginosa improves its resistance to the innate clearance mechanisms in the lung.     

Protease phenotypes of Pseudomonas aeruginosa isolated from patients with cystic fibrosis.

The protease phenotypes expressed by isolates of Pseudomonas aeruginosa from cystic fibrosis (CF) patients were evaluated. The majority of isolates tested produced elastase (65%) or alkaline protease (64%) or both. The mucoid phenotype expressed by many CF isolates of P. aeruginosa did not absolutely restrict the expression of protease activity, although a higher percentage of nonmucoid isolates was proteolytic. When isolates from CF patients chronically infected with P. aeruginosa were compared to isolates from CF patients colonized with this organism, both groups were found to contain comparable percentages of elastase-producing strains and mucoid strains. However, the group of isolates from colonized patients contained a higher percentage of strains producing alkaline protease and expressing general protease activity. In addition, the group of isolates from chronically infected patients contained more weakly proteolytic isolates than either the group from colonized CF patients or a group of isolates from pediatric patients without CF. These data suggest that protease production may be important in the initial colonization of the respiratory tract of CF patients by P. aeruginosa.     

Serotyping of Pseudomonas aeruginosa isolates from patients with cystic fibrosis of the pancreas.

Pseudomonas aeruginosa isolates (173) from 144 patients with cystic fibrosis (CF) of the pancreas in seven hospitals were serotyped with the agglutination systems of Homma (1974) and Fisher et al. (1969). The two systems were complementary. Strains from CF patients were much less likely to furnish a stable type on repetitive typing tests than strains from other patients. This was related to the frequent occurrence of mucoid P. aeruginosa strains. The 173 strains were divided among 11 Homma serotypes. A single Homma type (type 8) capable of mucoid growth comprised 104 (60%) CF strains. Eight serotypes were detected in 77 strains from 48 CF patients in one hospital; three strains were detected in one hospital CF unit; and two strains were detected in each of five hospital CF units. The CF serotype comprised from 50 to 93% of CF strians inthe seven hospitals. These P. aeruginosa strains dissociated in vivo as judged by mucoid and nonmucoid colonies on primary culture plates and continued to dissociate during subcultures. Both colony type were the same serotype. The tendency to regard colonial phenotypes (mucoid, nonmucoid, rough) as separate strians was erroneous. Repetitive typing with the two systems gave better results than a single system. The mucoid P. aeruginosa strain is probably spread from patient to patient, rather than acquiring its mucoid characteristic de novo in the CF patient. It is not known why the mucoid CF strain has a peculiar predilection for CF patients, nor why it generally loses the quality in culture but retains it indefinitely in the patient.     

Conversion of Pseudomonas aeruginosa to the phenotype characteristic of strains from patients with cystic fibrosis.

Isolates of Pseudomonas aeruginosa from cystic fibrosis patients are unusual; they are often susceptible to the bactericidal effect of human serum, have a rough lipopolysaccharide, and produce an exopolysaccharide that is responsible for the characteristic mucoid phenotype. In contrast, strains from the environment and from patients with other diseases usually have smooth lipopolysaccharide, do not produce very much mucoid exopolysaccharide, and are phenotypically nonmucoid. The predominance of mucoid strains of P. aeruginosa in infections of patients with cystic fibrosis has not been explained. In the lower airways, where P. aeruginosa persists in cystic fibrosis, nutrients for bacterial growth may be limited. We investigated whether growth of P. aeruginosa under conditions of suboptimal nutrition causes conversion to the characteristic cystic fibrosis phenotype. Ninety-two strains of P. aeruginosa were maintained for up to 90 days in a minimal medium with acetamide as the sole carbon source. In 56 (52%) of 107 cultures, isolates with rough lipopolysaccharide emerged, and in 20 (19%) of 104 nonmucoid cultures, mucoid isolates were recovered. Strains with rough lipopolysaccharide also were sensitive to the bactericidal effect of normal human serum. Under conditions of suboptimal nutrition in vitro, isolates of P. aeruginosa emerged that produced rough lipopolysaccharide and were mucoid, typical of many isolates from cystic fibrosis patients. This peculiar phenotype may arise as a consequence of nutritional limitation within the cystic fibrosis respiratory tract rather than from features unique to these strains of bacteria.     

Comparison of the outer membrane protein and lipopolysaccharide profiles of mucoid and nonmucoid Pseudomonas aeruginosa.

Laboratory-derived mucoid variants of Pseudomonas aeruginosa were selected by plating the standard PAO1 laboratory strain with bacteriophage. These mucoid variants formed two distinct groups of strains on the basis of phage typing. The first group had the same phage-typing pattern as the parent PAO1 strain, while the second group had a distinctly different phage-typing pattern. One strain from each group was assessed along with the parent PAO1 strain for its outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles by sodium dodecyl sulfate-gel electrophoresis followed by appropriate staining. The mucoid derivatives were found to differ from the parent PAO1 nonmucoid strain in having lost a high-molecular-weight LPS species. Furthermore, the reversion of the mucoid strains to the nonmucoid phenotype was accompanied by a return of the missing high-molecular-weight LPS species. No observable difference between the mucoid derivatives and the parent nonmucoid strain was noted in the OMP profiles. The opposite was found in the case of four isolates of mucoid P. aeruginosa from patients with cystic fibrosis. Two OMP bands (of approximately 55 and 25 kilodaltons) were present in the mucoid isolates but missing in their sister nonmucoid strains. In the case of the cystic fibrosis isolates, no difference in the LPS profiles within mucoid-nonmucoid pairs was noted.     

Biologic activities of antibodies to the neutral-polysaccharide component of the Pseudomonas aeruginosa lipopolysaccharide are blocked by O side chains and mucoid exopolysaccharide (alginate).

Virulent strains of Pseudomonas aeruginosa are either of a nonmucoid, lipopolysaccharide (LPS)-smooth or mucoid, LPS-rough phenotype, and immunity to these different variants is efficiently mediated by antibodies specific to O antigens or mucoid exopolysaccharide (also called alginate), respectively. In addition to O side chains and core polysaccharide components, the LPS of P. aeruginosa also contains neutral-polysaccharide components that express antigenic determinants common to many clinical isolates. We evaluated antibodies specific to neutral polysaccharides for the ability to mediate opsonic killing and protective immunity. Antibodies to these antigens mediated opsonic killing of poorly virulent nonmucoid LPS-rough isolates but not of isogenic strains with either a LPS-smooth or a mucoid phenotype. Antibodies to neutral-polysaccharide antigens also failed to protect neutropenic mice from challenge with modest doses of LPS-smooth P. aeruginosa strains (< 10(3) CFU per mouse), whereas O-antigen-specific antibodies were highly protective. Antibodies to neutral polysaccharides deposited significantly (P = 0.002) more C3 onto LPS-rough strains than did antibodies to O side chains, but this situation was reversed when isogenic LPS-smooth strains were tested. Given that protective immunity against P. aeruginosa must be directed against either nonmucoid LPS-smooth strains or mucoid LPS-rough strains, it appears that antibodies specific to neutral-polysaccharide antigens do not protect against P. aeruginosa infection. Lack of protection is likely due to the ability of both O side chains and mucoid exopolysaccharide (alginate) to interfere with the opsonic killing activity of neutral-polysaccharide-specific antibodies.     

Development of an enzyme-linked immunosorbent assay for studying Pseudomonas aeruginosa cell surface antigens.

An enzyme-linked immunosorbent assay for the measurement of antibodies directed against Pseudomonas aeruginosa cell surface antigens was developed. Formalin-killed whole cells of P. aeruginosa, adsorbed to polystyrene acrylic copolymer cuvettes, were used as immobilized antigens. Antisera to P. aeruginosa mucoid strain 144M and to its spontaneous nonmucoid derivative, 144NM, were raised in rabbits by immunization with Formalin-killed bacteria. By using this enzyme-linked immunosorbent assay, anti-144M serum was found to have a ca. 10-fold-higher antibody titer to 144M than did anti-144NM serum, suggesting that 144M may have either immunogenic determinants not present on 144NM or perhaps simply more antigenic determinants. In contrast, anti-144M and anti-144NM immune sera were found to have nearly identical antibody titers to 144NM, suggesting that these strains share many determinants. Anti-P. aeruginosa immune serum was found to contain Pseudomonas-specific antibodies as well as antibodies which cross-reacted with other gram-negative bacteria. Finally, absorption studies demonstrated that this assay can detect both LPS and non-LPS surface-exposed antigenic determinants. Thus, this whole bacterial cell enzyme-linked immunosorbent assay should prove useful in monitoring patient sera and secretions for potentially protective immunoglobulins directed at P. aeruginosa cell surface antigens.     

Functional role of mucoid exopolysaccharide (alginate) in antibiotic-induced and polymorphonuclear leukocyte-mediated killing of Pseudomonas aeruginosa.

We evaluated in vitro the functional role of mucoid exopolysaccharide (MEP) of Pseudomonas aeruginosa in blocking antibiotic-induced and polymorphonuclear leukocyte (PMN)-mediated pseudomonal killing. The serum-resistant P. aeruginosa isolates used were mucoid strain 144MR and its nonmucoid revertant, strain 144NM. By timed kill curves, early bacterial effects of amikacin against mucoid strain 144MR were substantially less than those observed with nonmucoid strain 144NM; this effect was reversible with enzymatic hydrolysis of MEP of strain 144MR by alginase. Also, early tobramycin uptake (15 to 30 min) by mucoid 144MR cells was less than that seen with nonmucoid strain 144NM; pretreatment of 144MR cells with alginase substantially enhanced early tobramycin uptake compared with untreated 144MR cells (P = 0.08). In strain 144NM (but not in strain 114MR) there was a notable postantibiotic leukocidal enhancement effect manifested by increased nonopsonic killing following brief exposure of these cells to supra-MIC amikacin; pretreatment of strain 144MR with alginase rendered these cells more susceptible to amikacin-induced postantibiotic leukocidal enhancement. Similarly, direct PMN-mediated nonopsonic killing of mucoid strain 144MR was significantly less than that observed with strain 144NM (P less than 0.05); pretreatment of 144MR cells with alginase rendered this strain equal to strain 144NM in susceptibility to nonopsonic killing. In addition, exogenous sodium alginate or extracted MEP of strain 144MR interfered with effective nonopsonic killing of strain 144NM by PMNs. Studies also indicated that mucoid strain 144MR was phagocytosed significantly less well than its nonmucoid mate (P less than 0.00001), an effect reversed by pretreatment of the mucoid cells with alginase. These data confirm that P. aeruginosa MEPs functionally decrease the uptake and early bactericidal effect of aminoglycosides in vitro and interfere with effective PMN-mediated nonopsonic phagocytosis and killing of mucoid strains.     

Comparison of agar diffusion methodologies for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of patients with cystic fibrosis (CF). Improved antimicrobial chemotherapy has significantly increased the life expectancy of these patients. However, accurate susceptibility testing of P. aeruginosa isolates from CF sputum may be difficult because the organisms are often mucoid and slow growing. This study of 597 CF isolates of P. aeruginosa examined the correlation of disk diffusion and Etest (AB BIODISK, Solna, Sweden) results with a reference broth microdilution method. The rates of interpretive errors for 12 commonly used antipseudomonal antimicrobials were determined. The disk diffusion method correlated well (zone diameter versus MIC) for all of the agents tested. However, for mucoid isolates, correlation coefficients (r values) for piperacillin, piperacillin-tazobactam, and meropenem were <0.80. The Etest correlation with reference broth microdilution results (MIC versus MIC) was acceptable for all of the agents tested, for both mucoid and nonmucoid isolates. Category interpretation errors were similar for the disk diffusion and Etest methods with 0.4 and 0.1%, respectively, very major errors (false susceptibility) and 1.1 and 2.2% major errors (false resistance). Overall, both agar diffusion methods appear to be broadly acceptable for routine clinical use in susceptibility testing of CF isolates of P. aeruginosa.     

Evaluation of MicroScan Autoscan for identification of Pseudomonas aeruginosa isolates from cystic fibrosis patients

Accurate identification of gram-negative bacilli from cystic fibrosis (CF) patients is essential. Only 57% (108 of 189) of nonmucoid strains and 40% (24 of 60) of mucoid strains were definitively identified as Pseudomonas aeruginosa with MicroScan Autoscan. Most common misidentifications were Pseudomonas fluorescens-Pseudomonas putida (i.e., the strain was either P. fluorescens or P. putida, but the system did not make the distinction and yielded the result P. fluorescens/putida) and Alcaligenes spp. Extending the incubation to 48 h improved identification, but 15% of isolates remained misidentified. The MicroScan Autoscan system cannot be recommended for the identification of P. aeruginosa isolates from CF patients.     

Expression and antimicrobial function of bactericidal permeability-increasing protein in cystic fibrosis patients

In cystic fibrosis (CF), the condition limiting the prognosis of affected children is the chronic obstructive lung disease accompanied by chronic and persistent infection with mostly mucoid strains of Pseudomonas aeruginosa. The majority of CF patients have antineutrophil cytoplasmic antibodies (ANCA) primarily directed against the bactericidal permeability-increasing protein (BPI) potentially interfering with antimicrobial effects of BPI. We analyzed the expression of BPI in the airways of patients with CF. In their sputum samples or bronchoalveolar lavage specimens, nearly all patients expressed BPI mRNA and protein, which were mainly products of neutrophil granulocytes as revealed by intracellular staining and subsequent flow cytometry. Repeated measurements revealed consistent individual BPI expression levels during several months quantitatively correlating with interleukin-8. In vitro, P. aeruginosa isolates from CF patients initiated the rapid release of BPI occurring independently of protein de novo syntheses. Furthermore, purified natural BPI as well as a 27-mer BPI-derived peptide displayed antimicrobial activity against even patient-derived mucoid P. aeruginosa strains and bacteria resistant against all antibiotics tested. Thus, BPI that is functionally active against mucoid P. aeruginosa strains is expressed in the airways of CF patients but may be hampered by autoantibodies, resulting in chronic infection.     

Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains

Twenty-six Pseudomonas aeruginosa strains from patients with cystic fibrosis were typed by the Fisher immunotyping scheme. Only 6 strains were agglutinated by a single typing serum, whereas 15 strains were agglutinated with more than one serum and 5 were not agglutinated by any serum. Neither the polyagglutinable nor the nonagglutinable strains were typable by hemagglutination inhibition or immunodiffusion, suggesting that these polyagglutinable strains did not express multiple serotype antigens, but were instead being agglutinated by antibody to nonserotype determinants. Four typable isolates were resistant to pooled normal human serum, whereas the 12 polyagglutinable and nonagglutinable isolates studied were very sensitive to normal human serum. The outer membranes of 16 strains were isolated and characterized. The data suggested, in general, strong conservation of outer membrane protein patterns. Lipopolysaccharides (LPS) were purified by a new technique which allowed isolation of both rough and smooth LPS in high yields. Three of four typable, serum-resistant strains examined had amounts of smooth, O-antigen-containing LPS equivalent to our laboratory wild type, P. aeruginosa PAO1 strain H103. In contrast, 10 of 12 polyagglutinable or nonagglutinable, serum-sensitive strains had very little or no smooth, O-antigen-containing LPS, and the other two contained less smooth LPS than our wild-type strain H103. In agreement with this data, five independent, rough, LPS O-antigen-deficient mutants of strain H103 were nontypable and serum sensitive. We suggest that the LPS defects described here represent a significant new property of many P. aeruginosa strains associated with cystic fibrosis.     

Pseudomonas aeruginosa adhesins for tracheobronchial mucin.

Mucins of the tracheobronchial tree are preferential sites for adherence and colonization by Pseudomonas aeruginosa. They possess specific receptors for this organism that have amino sugars as their principal constituents. Since mucins probably reflect the receptors on the cellular surfaces, we hypothesized that the bacterial adhesins previously shown to mediate the binding of P. aeruginosa to cells would also mediate bacterial binding to mucins. We therefore tested the roles of the exopolysaccharide from mucoid strains of P. aeruginosa and pili from nonmucoid strains to see whether they are indeed the adhesins for mucins. Using a microtiter plate assay of adherence to mucins, we demonstrated that the mucoid exopolysaccharide bound to mucins and enhanced the adherence of mucoid strains to this substance. Antibodies raised against the exopolysaccharide from a single mucoid strain inhibited the adherence of all mucoid strains tested. Purified pili from nonmucoid strains inhibited the binding of nonmucoid strains but not of mucoid strains. Inhibition of adherence by antibody to pili was quite specific, antibody being able to inhibit only the binding of the homologous nonmucoid strain. These data support our previous observations with tracheal cells, confirming the similarity of the adhesins for respiratory tract cells and the mucins which cover them.     

Whole, submandibular, and parotid saliva-mediated aggregation of Pseudomonas aeruginosa in cystic fibrosis.

The aggregation of mucoid and nonmucoid Pseudomonas aeruginosa by submandibular, parotid, and whole saliva from patients with cystic fibrosis (CF) and non-CF subjects was investigated. There were significant differences (P less than 0.01) in aggregation of mucoid and nonmucoid variants of P. aeruginosa by submandibular and whole saliva from CF patients and non-CF subjects. However, the differences in the parotid secretion were not as pronounced. Patients with CF who were colonized with P. aeruginosa demonstrated a significantly higher (P less than 0.05) percent aggregation of the mucoid variants by the submandibular secretion and of both mucoid and nonmucoid variants by whole saliva, compared with corresponding secretions from patients with CF not colonized with this pathogen. The parotid saliva aggregation activity was not markedly different for the two groups with CF. From patients with CF, whole saliva demonstrated a higher percent P. aeruginosa aggregation than did the submandibular saliva. In non-CF subjects, however, the percent aggregation of P. aeruginosa by submandibular saliva was higher than that by whole saliva. Our results indicate that the sero-mucous products of the submandibular gland have a more significant role in P. aeruginosa aggregation than the serous secreting parotid cells and that the submandibular secretion is possibly responsible for the differences in oral colonization by this pathogen in subjects with and without CF.     

Alginate--its role in neutrophil responses and signal transduction towards mucoid Pseudomonas aeruginosa bacteria.

Mucoid Pseudomonas aeruginosa bacteria impaired neutrophil functions, e.g. chemiluminescence response, and leukotriene formation to a significantly higher degree as compared to nonmucoid P. aeruginosa bacteria. To study the cell biological requirements for the different cellular response pattern by mucoid and nonmucoid (NM) P. aeruginosa bacteria, further experiments were performed with purified alginate, the mucoid exopolysaccharide of P. aeruginosa (MEP). In this regard the MEP (alginate) significantly reduced the zymosan-induced leukotriene B4 (LTB4) formation (from 40 +/- 7 to 2 +/- 4 ng). The chemiluminescence response induced by NM bacteria was abolished when the bacteria were precoated with the MEP. Mucoid and NM P. aeruginosa bacteria interacted with components of the cellular signal transduction pathway to a different degree. Mucoid bacteria induced a 2-fold enhanced GTPase activity but activated the protein kinase C (PKC) to a lesser degree than NM P. aeruginosa bacteria. Prior exposure of neutrophils to the MEP increased the sodium fluoride (NaF)-induced GTPase activity and guanylylimidodiphosphate binding [Gpp(NH)p] by approximately 60 and 30%, respectively. The phorbol myristic acid-induced PKC activation was inhibited by 30-40% in the presence of the MEP. However, the MEP by itself was inactive in all assay systems. Our results indicate that the MEP represents an important component which modulates neutrophil responses of mucoid as compared to NM P. aeruginosa bacteria, e.g. the chemiluminescence response, LTB4 generation, and the interaction with components (G proteins, PKC) of the signal transduction pathway.     

Inhibition of adherence of mucoid Pseudomonas aeruginosa by alginase, specific monoclonal antibodies, and antibiotics.

The adherence of pseudomonal species was investigated by using a newly developed radiometric dacron fiber microcolumn assay. Pseudomonas aeruginosa, P. stutzeri, and Xanthomonas maltophilia were more adherent (approximately 20%) than P. pseudomallei, P. fluorescens, and P. cepacia (approximately 10%). Mucoid strains of P. aeruginosa were consistently more adherent than nonmucoid strains (30% versus 20%). Alginase was shown to inhibit the adherence of mucoid but not nonmucoid P. aeruginosa. Monoclonal antibodies to alginate were also shown to inhibit the adherence of mucoid but not nonmucoid P. aeruginosa. In addition, antibiotics active against P. aeruginosa were shown to inhibit the adherence of both mucoid and nonmucoid strains. Furthermore, synergism between dyadic combinations of monoclonal antibodies and antibiotic (ciprofloxacin), as well as alginase and antibiotic, was also observed. These results indicate that bacterial alginate has an intrinsic role in the adherence of mucoid P. aeruginosa and may have evolved not only for protection against dehydration in the water and soil ecosystem of this bacterium, but also as a means of attaching to soil substrates in the same ecosystem to enhance survival. They also suggest that synergistic combinations of antibiotics with alginase or monoclonal antibodies to alginate may be of value in the therapy of some pseudomonal infections.     

Surfactant protein A and D differently regulate the immune response to nonmucoid Pseudomonas aeruginosa and its lipopolysaccharide

We investigated the role of the surfactant proteins (SPs) A and D in the pulmonary immune defense of nonmucoid strains of Pseudomonas aeruginosa, the most etiologic agents of nosocomial Pseudomonas pneumonia. We first examined the interactions of recombinant human SP-D dodecamers and purified natural or recombinant human SP-A with two smooth, and two rough, clinical isolates of nonmucoid P. aeruginosa. SP-D bound to all four isolates, but agglutinated only one rough and one smooth strain. SP-D functioned as an opsonin to enhance the uptake of all four strains by the human monocytic cell line Mono Mac 6 (MM6). SP-D also enhanced tumor necrosis factor-alpha secretion by MM6 cells in response to purified lipopolysaccharide (LPS) isolated from the rough, but not the smooth, strains. Although SP-A bound to all four strains, it did not cause bacterial aggregation or enhance uptake. It showed small but statistically significant inhibitory effects on the cytokine response of MM6 cells to one strain of smooth organisms, but did not significantly alter the response to purified LPS. This study in combination with previously published data strongly suggests that SP-D may play important roles in the local innate pulmonary defense against nonmucoid P. aeruginosa of diverse LPS phenotypes, and preferentially augments the cellular response to rough P. aeruginosa endotoxin.     

Production and characterisation of mouse monoclonal antibodies reactive with the lipopolysaccharide core of Pseudomonas aeruginosa.

Monoclonal antibodies (MAbs) to the core antigen region of lipopolysaccharide (LPS) of Pseudomonas aeruginosa were produced from mice immunised with whole cells of heat-killed rough mutants of Pseudomonas aeruginosa expressing partial or complete core LPS. MAbs were screened in an enzyme-linked immunosorbent assay (ELISA) against three different antigen cocktails: S-form LPS from three P. aeruginosa strains, R-form LPS from six P. aeruginosa strains and, as a negative control, R-form LPS from Salmonella typhimurium and Escherichia coli. Selected MAbs were subsequently screened against a range of extracted LPS and whole cells from both reference strains and clinical isolates of P. aeruginosa. The antibodies were also screened in ELISA against whole-cell antigens from other Pseudomonas spp. as well as strains of Haemophilus influenzae, Neisseria subflava and Staphylococcus aureus. Five MAbs reacting with the core component of P. aeruginosa LPS were finally selected. Two of these, MAbs 360.7 and 304.1.4, were particularly reactive in immunoblots against unsubstituted core LPS, including that from O-antigenic serotypes of P. aeruginosa. The MAbs also reacted with some of the other Pseudomonas spp., but not with P. cepacia or Xanthomonas (Pseudomonas) maltophilia. Cross-reactivity with whole cells from other bacterial species was minimal or not observed. Reactivity of MAbs with some Staph. aureus strains was observed, and binding to the protein A component was implicated. The reactivity of the MAbs was investigated further by flow cytometry and immunogold electronmicroscopy. The suitability of the MAbs for an immunological assay for detection of P. aeruginosa in respiratory secretions from CF patients is discussed.     

Evaluation of Reference Dilution Test Methods for Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa Strains Isolated from Patients with Cystic Fibrosis

The development of multidrug-resistant Pseudomonas aeruginosa in patients with cystic fibrosis (CF) is most likely a consequence of increasing life expectancy and more prolonged exposure to antibiotics. The optimal method for antibiotic susceptibility testing of CF strains, particularly mucoid P. aeruginosa strains, is unknown. Antimicrobial susceptibilities of 48 CF strains (25 mucoid) and 50 non-CF strains to 12 anti-Pseudomonas agents were tested by both agar dilution and commercially custom-prepared broth microdilution plates (PML Microbiologicals, Portland, Oreg.) in three laboratories simultaneously to determine if broth microdilution could substitute for agar dilution as the reference method in subsequent studies. Comparison of MICs generated by agar dilution and broth microdilution demonstrated correlation coefficients (r) exceeding 0.85 for all agents tested; correlation was excellent for aminoglycosides (r >/= 0.92) and very good for beta-lactam agents including agents paired with a beta-lactamase inhibitor (r >/= 0.87) and for ciprofloxacin (r = 0.86). Correlation was not improved by 48-h readings, but correlation between 24- and 48-h readings ranged between 0.91 and 0.98 for both methods. Interlaboratory variations were minimal, as the percentage of acceptable variations was 94% for both methods, and serious discords were infrequent (<2% of comparisons). However, CF strains were more likely to have serious discords than were non-CF strains (P < 0. 0001), although mucoid strains were not more likely to have serious discords than were nonmucoid strains. In this study, MICs determined by custom-prepared broth microdilution compared favorably with MICs determined by agar dilution. Thus, this broth microdilution assay can serve as a reference method and facilitate future studies to determine the optimal method for antibiotic susceptibility testing of CF strains.