AP-PCR typing of Pseudomonas aeruginosa isolated from patients with cystic fibrosis

Arbitrarily Primed Polymerase Chain Reaction has been used for an epidemiological evaluation of 42 strains of P. aeruginosa isolated from nine cystic fibrosis patients during a three-year investigation period. The resistance patterns of the same strains have also been evaluated. The AP-PCR type fingerprinting was perfomed with primers 10514 and 208. Resistance was evaluated by the Minimal Inhibitory Concentration method. With 10514 eleven different genotypes could be evidenced, while with 208 only five of them could be detected. During the investigation period patients were always colonised by the same genotype. A possible correlation between resistance pattern and genotype with both primers has shown, within the same patient, a correspondence of about 20% for 10514 and a correspondence of only 10% for 208. Patients are colonised by one or two strains of P. aeruginosa and there is no relation between genotype and resistance pattern.     

Serum sensitivity of Burkholderia (Pseudomonas) cepacia isolates from patients with cystic fibrosis

Abstract Bacterial strains which are sensitive to the bactericidal activity of serum are generally considered to be less virulent than serum-resistant strains and are seldom associated with bacteraemia. Burkholderia ( Pseudomonas ) cepacia is an important pathogen in cystic fibrosis and is associated with rapid fatal pulmonary decline and bacteraemia in 20% of colonised patients. In this study 19 isolates of B. cepacia expressing either rough or smooth LPS were investigated to determine the degree of serum sensitivity. Strains expressing rough-LPS were serum-sensitive: these included a highly transmissible strain of B. cepacia isolated from approximately 50 cystic fibrosis patients attending various U.K. regional centres and associated with cases of bacteraemia.     

Siderophore synthesis by mucoid Pseudomonas aeruginosa strains isolated from cystic fibrosis patients.

Nonmucoid Pseudomonas aeruginosa responds to iron deprivation by synthesizing the siderophores pyochelin and pyoverdine. When grown in iron-deficient medium, six mucoid P. aeruginosa strains isolated from cystic fibrosis patients synthesized copious amounts of the exopolysaccharide alginate. A procedure that eliminated the interference of alginate was developed so that siderophores could be extracted from the growth medium. All six isolates were then noted to produce both pyoverdine and pyochelin. This report thus confirms that mucoid P. aeruginosa, like its nonmucoid counterparts, elicits the siderophores commonly cited as those of the microbe.     

Interbacterial adhesion between Pseudomonas aeruginosa and indigenous oral bacteria isolated from patients with cystic fibrosis

Interbacterial adhesion between strains of Pseudomonas aeruginosa and strains of indigenous oral bacteria, both of which were isolated from the oral cavity of cystic fibrosis patients, was investigated by the phenomenon of the coaggregation reaction. A total of 22 strains of P. aeruginosa were isolated from the oral cavity of 17 patients and examined for their abilities to coaggregate with 5 strains each of Streptococcus sanguis, Streptococcus mitis, Actinomyces viscosus, and Actinomyces naeslundii. Coaggregation reactions were common between these oral bacteria and both the mucoid and nonmucoid variants of P. aeruginosa. All strains of P. aeruginosa were also able to agglutinate neuraminidase-treated or untreated human erythrocytes of blood types A, B, and O. Positive coaggregation reactions were further characterized by determining the effects of several sugars, and of heat and protease treatments of the bacteria. None of the coaggregtion reactions were inhibited by 0.05 M lactose, galactose, glucose, fucose, or mannose. All coaggregation reactions were dependent upon heat- and protease-sensitive components of the Pseudomonas. Thus, the interbacterial adhesions between P. aeruginosa and the oral bacteria studied appears to involve adhesins on the Pseudomonas cell, which bind to complementary receptors, on the cell surfaces of oral bacteria. The apparent prevalence and diversity of interbacterial adhesions between P. aeruginosa strains originating from the oral cavity of cystic fibrosis patients and strains of the indigenous oral bacteria suggest that some of these reactions may affect the extent to which P. aeruginosa colonizes in the oral cavity of cystic fibrosis patients, and thereby, influence susceptibility of the host to infection.     

Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.

Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.     

Adherence ability of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis to two different epithelial cell lines

The ability of 59 wild-type strains of Pseudomonas aeruginosa to adhere to the HeLa and Buffalo Green Monkey Kidney (BGMK) cells was investigated. Twenty strains were isolated from sputa of cystic fibrosis patients, while 19 strains were isolated from tracheal aspirates and 20 from bronchial secretions of patients without cystic fibrosis, and they were used as a control group of strains. The statistically significant difference between adherence ability of strains was observed (p < 0.01). While most of the tracheal and bronchial isolates were hyperadhesive (51-110 bacteria per cell) most of the cystic fibrosis isolates adhered poorly to the HeLa and BGMK cells (1-10 bacteria per cell). The bacterial binding to the cells was blocked when bacteria were incubated at 80 degrees C for 20 min before the adherence assay. These results indicate that alginate is not involved in the adherence of P. aeruginosa to the used epithelial cell lines, and, because of that, mucoid strains isolated from persistently colonized cystic fibrosis patients showed poor adherence ability.     

Pseudomonas aeruginosa antibody detection in cystic fibrosis patients.

Chronic respiratory infection due to Pseudomonas aeruginosa remains the most important prognostic factor in cystic fibrosis patients. One method to lengthen the patient's life is to extend the initial state of the illness with an early diagnosis, before Ps. aeruginosa infection becomes chronic. Often this is difficult because of the young age of the patients. This study tested an immunoenzymatic system to evaluate antibody response against three Ps. aeruginosa purified antigens, alkaline protease, elastase and exotoxin A. We studied 40 patients with cystic fibrosis, 20 affected and 20 unaffected by apparent Ps. aeruginosa infection, also from the bacteriological point of view. Serological and bacteriological results were compared for each patient and showed that serological screening can be useful in young subjects, who often have no bacteriological evidence of Ps. aeruginosa colonization.     

Long-term preservation of strains of Burkholderia cepacia, Pseudomonas spp. and Stenotrophomonas maltophilia isolated from patients with cystic fibrosis

Long-term preservation methods are important in the maintenance of bacteria for downstream research applications. Most clinical laboratories have only limited resources for archiving isolates and therefore require cost-effective and simple methods. An effective and cheap storage method using debrinated blood and maintenance at -80 degrees C is described.     

Genotypic and phenotypic relationships in Burkholderia cepacia isolated from cystic fibrosis patients and the environment

Twenty-one strains of Burkholderia cepacia isolated from the environment, and 21 clinical strains isolated principally from sputum of cystic fibrosis (CF) patients, were characterized genotypically by macrorestriction analysis (genome fingerprinting) and PCR ribotyping, and phenotypically by susceptibility to antibiotics and the ability to macerate onion tissue. The plasmid content of the strains was also investigated. Environmental isolates showed a high degree of genetic variability, all strains differing from both one another and the CF isolates. The CF isolates were less variable, with common strains found in patients attending three geographically distinct CF centres. Phenotypic variation was found both within and between CF and environmental strains. Generally, CF isolates displayed higher levels of antibiotic resistance, while the ability to macerate onion tissue was more prevalent amongst environmental isolates. Plasmids were more frequently found in CF isolates, but were of similar size in both groups of strains. Such variability is not surprising in view of the existence of multiple genomovars within the B. cepacia complex.     

Characterization by pyocine typing and serotyping of oral and sputum strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients

Oral and sputum isolates of Pseudomonas aeruginosa in patients with cystic fibrosis were investigated. Of the 17 patients studied, 12 patients (71%) yielded both mucoid and nonmucoid variants of Pseudomonas aeruginosa from sputum and (or) various oral ecological sites, such as buccal mucosa, tongue dorsum, dental plaques, and saliva. A total of 51 strains of mucoid and nonmucoid Pseudomonas aeruginosa were isolated from these patients and were phenotypically characterized by both pyocine typing and serotyping. Five patients (42%) were colonized or infected by a single strain of Pseudomonas aeruginosa, whereas 7 patients (58%) were cocolonized or coinfected by two or more phenotypically different strains of Pseudomonas aeruginosa. To understand the mechanisms involved in Pseudomonas aeruginosa colonization, it may be necessary to identify multiple isolates of Pseudomonas aeruginosa not only from the sputum but also from the various oral ecological sites and to further explore the role of the oral cavity in this colonization.     

An intragenic deletion in pilQ leads to nonpiliation of a Pseudomonas aeruginosa strain isolated from cystic fibrosis lung

Deficient motility is one of the characteristic hallmarks observed in Pseudomonas aeruginosa strains that chronically colonize the lungs of cystic fibrosis (CF) patients. Pseudomonas aeruginosa TB is a nonpiliated CF isolate known to be defective in twitching motility. Complementation confirmed a direct link of this phenotype to an intragenic out-of-frame deletion in pilQ (PA5040). Sequence alignment of pilQ derived from TB vs. PAO1 suggests that close direct repeats framing the deletion site may have triggered this mutation. This type of mutation could play a role in the emergence of pathoadaptive mutations of P. aeruginosa in the CF lung habitat.     

Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: Assessment of cross-reactivity with Pseudomonas aeruginosa

Abstract Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa . The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa . The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. This study suggests that there is a specific anti- B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa . Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS.     

Outer membrane antigens of mucoid Pseudomonas aeruginosa isolated directly from the sputum of a cystic fibrosis patient

Abstract The antigenicity of the outer membrane components of mucoid Pseudomonas aeruginosa directly isolated from the sputum of a cystic fibrosis patient and those of the same isolate cultivated under iron-depleted conditions in the presence of sub-in-hibitory concentrations of piperacillin and/or tobramycin was investigated by immunoblotting using the patient's own serum. The results indicated that iron-regulated membrane proteins as well as other major outer membrane proteins were antigenic and recognised by the patient's serum. The antibiotics used profoundly influenced the surface antigen pattern.     

Anaerobic culture conditions favor biofilm-like phenotypes in Pseudomonas aeruginosa isolates from patients with cystic fibrosis

Pseudomonas aeruginosa causes chronic infections in the lungs of cystic fibrosis (CF) individuals and remains the leading cause of morbidity and mortality associated with the disease. Biofilm growth and phenotypic diversification are factors thought to contribute to this organism's persistence. Most studies have focused on laboratory isolates such as strain PAO1, and there are relatively few reports characterizing the properties of CF strains, especially under decreased oxygen conditions such as occur in the CF lung. This study compared the phenotypic and functional properties of P. aeruginosa from chronically infected CF adults with those of strain PAO1 and other clinical non-CF isolates under aerobic and anaerobic culture conditions. The CF isolates overall displayed a reduced ability to form biofilms in standard in vitro short-term models. They also grew more slowly in culture, and exhibited decreased adherence to glass and decreased motilities (swimming, swarming and twitching). All of these characteristics were markedly accentuated by anaerobic growth conditions. Moreover, the CF strain phenotypes were not readily reversed by culture manipulations designed to encourage planktonic growth. The CF strains were thus inherently different from strain PAO1 and most of the other non-CF clinical P. aeruginosa isolates tested. In vitro models used to research CF isolate biofilm growth need to take the above properties of these strains into account.     

Differential binding of Pseudomonas aeruginosa to normal and cystic fibrosis tracheobronchial mucins

Pseudomonas aeruginosa infection is a leading cause of deteriorationof pulmonary function in patients with cystic fibrosis (CF).The interaction of the bacterium with CF and non-CF tracheobronchialmucins was examined to understand the biochemical basis forthe high susceptibility of the lungs of CF patients to infectionby P.aeruginosa. The binding of radiolabelled bacteria to puremucins in solid-phase assays was not significantly above non-specificbinding to various blocking agents, such as bovine serum albumin,Tween 20, milk powder and polyvinyl pyrrolidine. Further, therewas a tendency for the bacteria to be excluded from plasticwells and membranes coated with mucin. Therefore, an indirectapproach involving the binding of radiolabelled P.aeruginosato asialo GM₁ ganglioside, the putative receptor for the bacteriaon tracheal cells, was used to compare the interaction of CFand non-CF mucins with the bacteria. Highly purified preparationsof CF mucin were consistently better inhibitors of the bindingof the bacteria to asialo GM₁ ganglioside than non-CF mucinpreparations. In the case of the binding of a stable mucoidstrain, the difference was statistically significant (P <0.001) at all concentrations of mucin tested. For the non-mucoidstrain, the difference was significant only at the higher concentrations.Of the saccharides tested similarly, sialyl lactose and theoligosaccharide portion of asialo GM₁ were found to be goodinhibitors. The increased binding of the bacteria to CF mucinwas further confirmed by a solution binding assay in which thebinding of ¹²⁵I-labelled mucin to unlabelled bacteria was determined.The binding of the bacteria to labelled CF and non-CF mucincould be inhibited by an excess of unlabelled human tracheobronchialmucin, but not by unrelated mucins, hyaluronic acid, alginicacid, bovine serum albumin and tetramethyl urea. The higherbinding of CF mucin, particularly to the mucoid strain of P.aeruginosa,is interesting and provides a model system to further investigatethe biochemical parameters of the interaction. asialo GM₁ ganglioside cystic fibrosis Pseudomonas aeruginosa respiratory mucins saccharide inhibitors.