Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis.

Pseudomonas aeruginosa isolates recovered from chronically colonized patients with cystic fibrosis (CF) are phenotypically different from those collected from other patients or from the environment. To assess whether alterations in motility, mucoidy, and serum susceptibility represented an adaptation to chronic infection or replacement by a new strain, sequential P. aeruginosa isolates of known phenotype collected from 20 CF patients were typed by random amplified polymorphic DNA (RAPD) analysis. A total of 35 RAPD strain types were found among 385 isolates from 20 patients, and only two patients had P. aeruginosa strains of the same RAPD fingerprint. Eight strain pairs representative of the first eight RAPD types were also analyzed by SpeI macrorestriction followed by pulsed-field gel electrophoresis (PFGE); the strain types found by both fingerprinting techniques correlated exactly. In 11 of 20 patients, the RAPD types of serial P. aeruginosa isolates remained stable despite alterations in isolate motility, colonial morphology, and lipopolysaccharide phenotype. However, in isolates collected from one CF patient, a single band change in RAPD fingerprint and CeuI PFGE profile correlated with the appearance of an RpoN mutant phenotype, suggesting that the altered phenotype may have been due to a stable genomic rearrangement. Secretion of mucoid exopolysaccharide, loss of expression of RpoN-dependent surface factors, and acquisition of a serum-susceptible phenotype in P. aeruginosa appear to evolve during chronic colonization in CF patients from specific adaptation to infection rather than from acquisition of new bacterial strains.     

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.     

Heterogeneity of Pseudomonas aeruginosa in Brazilian Cystic Fibrosis Patients

The aim of this study was to assess the diversity and genomic variability of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients being treated at a university hospital in Brazil. Ninety-seven isolates of P. aeruginosa from 43 CF patients were characterized by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE) and tested for susceptibility to 20 antimicrobial agents by broth microdilution. It was possible to evaluate single isolates from 20 patients and multiple isolates (two to seven) from 23 patients collected during a 22-month period. Among all of the unrelated patients, we detected only one pair of patients sharing a common strain. Among the 77 isolates from 23 patients who had multiple isolates analyzed, we identified 37 major types by PFGE, and five different colonization patterns were recognized. The isolates were susceptible to several antimicrobial agents, although consecutive isolates from the same patient may display differences in their susceptibilities. Mucoid isolates were more resistant (P < 0.001) than nonmucoid isolates to five antibiotics. Our results indicate that CF patients remain colonized by more than one strain of P. aeruginosa for long periods of time. In addition, the finding of several different genotypes in the same patient suggests that the colonizing strain may occasionally be replaced.     

Genome macrorestriction analysis of diversity and variability of Pseudomonas aeruginosa strains infecting cystic fibrosis patients.

Genome macrorestriction fingerprinting with XbaI and DraI was used to analyze the relatedness of 166 Pseudomonas aeruginosa isolates collected from 31 cystic fibrosis patients over a 1- to 20-month period and to correlate their genotype with patterns of resistance to 14 antimicrobial agents. Quantitative comparison of intra- and interpatient similarities of P. aeruginosa macrorestriction patterns disclosed two discrete ranges that clearly discriminated subclonal variation (> 80% relatedness) and clonal diversity (10 to 70% relatedness). Cloning-derived mutants exhibited up to 20% divergence of genomic macrorestriction patterns during the course of chronic colonization of individual patients. Change of susceptibility to multiple antimicrobial agents developed in 50% of sequential pairs of isolates from individual patients. Only 19% of these susceptibility changes were attributable to strain substitution, while the majority (56%) of resistance changes were associated with minor genomic variations of a persistent strain. Sixty-six percent of patients harbored one strain, and 33% carried two strains. Three common strains colonized 5 (28%) of 18 patients attending a cystic fibrosis clinic, and another two strains colonized two patient pairs (31%) of 13 patients staying at a rehabilitation center, suggesting potential cross-infection in these settings. By indexing regional polymorphisms throughout the chromosome structure, macrorestriction analysis can monitor subclonal evolution of P. aeruginosa and identify isogenic resistance mutants. Quantitative macrorestriction fingerprinting enables discrimination between clonal variants and clones of distinct origins and should therefore provide a reliable tool for investigating the mode of acquisition of P. aeruginosa in cystic fibrosis patients.     

Nonmotility and phagocytic resistance of Pseudomonas aeruginosa isolates from chronically colonized patients with cystic fibrosis.

Although Pseudomonas aeruginosa chronically colonizes most older patients with cystic fibrosis (CF), bacterial features responsible for its persistence are understood poorly. We observed that many P. aeruginosa isolates from chronically colonized patients were nonmotile and resistant to phagocytosis by macrophages. P. aeruginosa isolates were collected from 20 CF patients for up to 10 years. Isolates from early colonization were highly motile and expressed both flagellin and pilin. However, many isolates from chronically colonized patients lacked flagellin expression and were nonmotile; a total of 1,030 P. aeruginosa CF isolates were examined, of which 39% were nonmotile. Moreover, sequential isolates recovered from several of the CF patients were consistently nonmotile for up to 10 years. Lack of motility was rare among environmental isolates (1.4%) and other clinical isolates (3.7%) of P. aeruginosa examined. Partial complementation of motility in nonmotile P. aeruginosa isolates was achieved by introduction of extra copies of the rpoN locus carried on plasmid pPT212, indicating that the alternate sigma factor, RpoN, may be involved in the coordinate regulation of virulence factors during CF infection. We hypothesize that the nonmotile phenotype may provide P. aeruginosa a survival advantage in chronic CF infection by enabling it to resist phagocytosis and conserve energy.     

Polysaccharide surface antigens expressed by nonmucoid isolates of Pseudomonas aeruginosa from cystic fibrosis patients.

We tested nonmucoid Pseudomonas aeruginosa isolates obtained from cystic fibrosis (CF) patients for the expression of lipopolysaccharide (LPS) serotype antigens, serum sensitivity, and production of mucoid exopolysaccharide (MEP). When all nonmucoid isolates were compared with a set of random mucoid isolates, 20 of 52 (38%) nonmucoid isolates were typable and serum resistant, compared with 13 of 51 (24%) mucoid isolates (P = 0.16 by chi-square analysis). However, nonmucoid strains from CF patients colonized only with nonmucoid strains were more frequently typable and serum resistant (67%) than were nonmucoid isolates from patients cocolonized with mucoid strains (31%) (P = 0.012, Fisher exact test). An inhibition enzyme-linked immunosorbent assay done with bacterial extracts, a direct-whole-cell enzyme-linked immunosorbent assay done with affinity-purified antibody to MEP, and immune electron microscopy all demonstrated production of MEP by all nonmucoid P. aeruginosa isolates tested, including nonmucoid revertants of mucoid strains. No other bacterial species tested positive in these assays. These findings suggest that MEP is produced by all P. aeruginosa isolates obtained from CF patients, that the initial colonizing nonmucoid strains produce a smooth LPS, and that once LPS-rough, mucoid strains appear in the sputum, the predominant LPS phenotype is rough regardless of colony morphology.     

Genome fingerprinting of Pseudomonas aeruginosa indicates colonization of cystic fibrosis siblings with closely related strains

The epidemiology of Pseudomonas aeruginosa infection at a cystic fibrosis (CF) center was monitored over a 3-year period. A total of 835 isolates from 72 unrelated patients and 22 siblings with CF were analyzed by genome fingerprinting and serotyping, bacteriophage typing, and pyocin typing. For genome fingerprinting, bacterial chromosomes were digested with one of the restriction endonucleases SpeI, DraI, XbaI, SspI, and NheI, which cut only rarely, and subsequently separated by field inversion gel electrophoresis. The physical genome analysis allowed us to classify P. aeruginosa strains in terms of DNA relatedness. Related strains differed by fewer than six DraI bands in the fingerprint, whereas unrelated strains differed by more than 20 DraI bands. All unrelated CF patients were colonized with different strains. The absence of a nosocomial spread of organisms at the CF center was attributed to the strict hygiene measures observed at the hospital. CF siblings were harboring either identical or closely related strains; transmission within the family is thought to be the most likely cause.     

Genome macrorestriction analysis of sequential Pseudomonas aeruginosa isolates from bronchiectasis patients without cystic fibrosis.

The respiratory tracts of bronchiectasis patients may be persistently colonized with Pseudomonas aeruginosa, despite intensive chemotherapy. The organism may undergo phenotypic changes in these patients, providing misleading typing results by conventional methods. We prospectively studied eight bronchiectasis patients without cystic fibrosis over a period of 1 year. A high microbial load of P. aeruginosa was found in 70% of sputum samples collected. Of these, 55 sequential P. aeruginosa isolates were characterized by a genotyping method, pulsed-field gel electrophoresis, to overcome the problem of differentiating the P. aeruginosa strains during chemotherapy. Genome macrorestriction fingerprinting patterns were analyzed after digestion with XbaI restriction endonuclease. Of the eight patients, six harbored a single dominant strain of P. aeruginosa, with an intrapatient macrorestriction similarity pattern range of 96 to 100%. The other two patients were infected with mixed bacterial isolates including P. aeruginosa. However, diversity was observed in the P. aeruginosa isolates from all eight patients, with a relatedness of only 55 to 65%. The study further strengthens the fact that pulsed-field gel electrophoresis can be used efficiently and effectively to differentiate P. aeruginosa strains in bronchiectasis patients without cystic fibrosis.     

Breakpoints for predicting Pseudomonas aeruginosa susceptibility to inhaled tobramycin in cystic fibrosis patients: use of high-range Etest strips

Inhaled administration of tobramycin assures high concentrations in cystic fibrotic lungs, improving the therapeutic ratio over that of parenteral tobramycin levels, particularly against Pseudomonas aeruginosa. Conventional Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) breakpoints only consider parenteral levels and do not take into account these high antimicrobial concentrations. The Spanish Antibiogram Committee (The MENSURA Group) has tentatively defined specific breakpoint values for inhaled tobramycin when testing P. aeruginosa isolates from cystic fibrosis (CF) patients (susceptible, < or =64 microg/ml; resistant, > or =128 microg/ml). The antimicrobial susceptibilities of 206 prospectively collected CF P. aeruginosa isolates were determined by the reference agar dilution method. For tobramycin, the performance of high range tobramycin Etest strips (AB Biodisk, Solna, Sweden) and conventional tobramycin disks were assessed with the same collection. Applying MENSURA proposed breakpoints, 95.1% of the strains were categorized as susceptible to tobramycin, either using agar dilution or Etest high-range strips (99% categorical agreement between both methods). With CLSI breakpoints, susceptibility rates decreased to 79.1 and 81.1% for agar dilution and Etest strips, respectively (83.5% categorical agreement). Minor, major, and very major errors for Etest strips (CLSI criteria) were 13.6, 1.2, and 14.8%, respectively. Upon applying the new proposed criteria for inhaled tobramycin, only one major and one very major error were observed with Etest strips. Whenever inhaled tobramycin is considered for therapy, we suggest that P. aeruginosa strains from CF patients categorized as intermediate or resistant to tobramycin according to the CLSI criteria should be retested with high-range Etest strips and recategorized using MENSURA interpretive criteria. CLSI breakpoints should still be followed when intravenous tobramycin is used in CF patients, particularly during the course of exacerbations.     

Genotypic analysis of Burkholderia cepacia isolates from 13 French cystic fibrosis centers.

Burkholderia cepacia has been involved in outbreaks of pulmonary infection among patients with cystic fibrosis (CF), and the spread of a highly transmissible clone has been reported throughout the United Kingdom and Canada. These data prompted a DNA-based typing study of the strains recovered in French CF centers. Ninety-five isolates recovered from 71 patients attending 13 CF centers in 9 regions of France were characterized by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Twenty-one genotypes were identified among the 95 isolates, and the results of RAPD and PFGE were concordant for 89 isolates (94%). Cross-colonization was demonstrated in 7 of the 13 CF centers. The investigation of serial isolates showed that most chronically colonized patients harbored a single B. cepacia strain. A geographically clustered distribution of B. cepacia genotypes was observed, except for one genotype, which was detected in four regions but was proven to be different from the genotype of the British-Canadian highly transmissible strain. The present study confirms the ability of B. cepacia to spread among CF communities in France and the importance of epidemiological surveys in the institution of prevention policies.     

Serotypes and Antibiotic Susceptibilities of Pseudomonas aeruginosa Isolates from Single Sputa of Cystic Fibrosis Patients

A phenotypic characterization of Pseudomonas aeruginosa from single sputum samples of 21 typical cystic fibrosis patients indicated a high frequency of heterogeneity among isolates on the basis of differences in antibiotic resistance, colony morphology, pigmentation, and serotype. Two or more isolates with different but stable susceptibilities to carbenicillin, gentamycin, streptomycin, tetracycline, chloramphenicol, and sulfamethoxazole plus trimethoprim were detected in 38% of the sputa. Differences generally were independent of the mucoid state of the strain. O-antigen group determination with the Difco typing set showed that two or more serologically distinct strains were present in 10/21 sputum specimens. Nonmucoid derivatives of mucoid isolates almost always retained both the antibiotic susceptibilities and serotype of their parent strain. These data suggest that cystic fibrosis patients may be cocolonized/coinfected by different strains of P. aeruginosa more frequently than generally believed. Alternatively, phenotypically distinct strains from a single patient might arise as phenotypic dissociants from a single infecting strain. Because of the frequency and multiplicity of phenotypically distinct P. aeruginosa isolates which we obtained from our cystic fibrosis patients, it is important to select multiple isolates from sputum cultures for antimicrobial susceptibility testing so as to assess adequately the susceptibility of this organism to antibiotic therapy in cystic fibrosis. We recommend that several colonies of each distinguishable colony type of P. aeruginosa be pooled for the antibiogram.     

Comparison of arbitrarily primed PCR and macrorestriction (pulsed-field gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients.

Arbitrarily primed PCR fingerprinting was carried out on 43 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. Seventeen major groups of strains that coincided with groups also distinguished by macrorestriction (pulsed-field gel electrophoresis) typing were identified. Our results illustrated that a CF patient can carry more than one strain and can carry a given strain for long periods of time and that strains can evolve by changes in drug resistance or other phenotypic traits during long-term colonization. The arbitrarily primed PCR method is recommended for first-pass screening of P. aeruginosa isolates from CF patients, especially when many strains are to be typed, because of its sensitivity and efficiency.     

Comparison of three molecular techniques for typing Pseudomonas aeruginosa isolates in sputum samples from patients with cystic fibrosis

Monitoring the emergence and transmission of Pseudomonas aeruginosa strains among cystic fibrosis (CF) patients is important for infection control in CF centers internationally. A recently developed multilocus sequence typing (MLST) scheme is used for epidemiologic analyses of P. aeruginosa outbreaks; however, little is known about its suitability for isolates from CF patients compared with that of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). As part of a prevalence study of P. aeruginosa strains in Australian CF clinics, we compared the discriminatory power and concordance of ERIC-PCR, PFGE, and MLST among 93 CF sputum and 11 control P. aeruginosa isolates. PFGE and MLST analyses were also performed on 30 paired isolates collected 85 to 354 days apart from 30 patients attending two CF centers separated by 3,600 kilometers in order to detect within-host evolution. Each of the three methods displayed high levels of concordance and discrimination; however, overall lower discrimination was seen with ERIC-PCR than with MLST and PFGE. Analysis of the 50 ERIC-PCR types yielded 54 PFGE types, which were related by ≤ 6 band differences, and 59 sequence types, which were classified into 7 BURST groups and 42 singletons. MLST also proved useful for detecting novel and known strains and for inferring relatedness among unique PFGE types. However, 47% of the paired isolates produced PFGE patterns that within 1 year differed by one to five bands, whereas with MLST all paired isolates remained identical. MLST thus represents a categorical analysis tool with resolving power similar to that of PFGE for typing P. aeruginosa. Its focus on highly conserved housekeeping genes is particularly suited for long-term clinical monitoring and detecting novel strains.     

A novel mucin-sulphatase activity found in Burkholderia cepacia and Pseudomonas aeruginosa.

Lung infections due to Burkholderia cepacia and Pseudomonas aeruginosa in patients with cystic fibrosis (CF) are common, are associated with respiratory morbidity and are a cause of mortality. Respiratory mucin in CF patients is highly sulphated, which increases its resistance to bacterial degradation. Desulphation increases the susceptibility of mucin to degradation by bacterial glycosidases and proteinases, and subsequent deglycosylation may facilitate bacterial colonisation by increasing available substrates and binding sites. This study determined whether clinical and environmental strains of B. cepacia and P. aeruginosa had the ability to desulphate mucin. Mucin-sulphatase activity was tested by incubating bacterial cell suspensions with 35S-sulphated mucins purified from LS174T and HT29-MTX human colon carcinoma cell lines. These mucins were also used to test for differences in substrate specificities. Mucin-sulphatase activity was detected in all nine B. cepacia strains and in four of six P. aeruginosa strains. There was strain variability in the level of mucin-sulphatase activity. Aryl-sulphatase activities of Pseudomonas isolates (determined with methylumbelliferyl sulphate) were c. 20-fold higher than those of B. cepacia strains, and were independent of mucin-sulphatase activity. This is the first report to demonstrate desulphation of mucin by B. cepacia and P. aeruginosa. It is concluded that B. cepacia and P. aeruginosa produce one or more cell-bound glycosulphatase(s), in addition to aryl-sulphatase activity. Mucin-sulphatase activity of B. cepacia and P. aeruginosa may contribute to their association with airway infections in patients with cystic fibrosis.     

Detection by enzyme-linked immunosorbent assays of antibody specific for Pseudomonas proteases and exotoxin A in sera from cystic fibrosis patients.

Enzyme-linked immunosorbent assays were developed to measure serum antibody specific for Pseudomonas elastase, alkaline protease, and exotoxin A. Antibody responses to each Pseudomonas antigen were measured in cystic fibrosis (CF) patients who were not colonized with Pseudomonas aeruginosa, in those who were colonized, in those who were chronically infected with this organism, and in control subjects. Antibody levels for each antigen in the colonized and infected CF patients were higher than levels in uncolonized CF patients or non-CF control subjects. The antibody responses to elastase were similar in patients of the colonized and infected groups. However, infected CF patients had significantly elevated levels of antibody to exotoxin A (P less than 0.01) and alkaline protease (P less than 0.05) when compared with patients simply colonized with P. aeruginosa. These findings confirm that Pseudomonas alkaline protease, elastase, and exotoxin A are produced by Pseudomonas strains which colonize and infect CF patients. As an adjunct to established procedures (X-ray, microbiological culture, etc.), the antitoxin and anti-protease enzyme-linked immunosorbent assays may be clinically useful tests for differentiating colonized CF patients from those who have more severe Pseudomonas pulmonary infections.     

Phenotypic and genotypic characterization of Pseudomonas aeruginosa from cystic fibrosis patients

Pseudomonas aeruginosa accounts for about one half of all pulmonary infections of cystic fibrosis (CF) patients. In this study, we analyzed 135 P. aeruginosa strains isolated from the expectorations of 55 CF adult patients attending a CF referral center over a period of five years. We assessed the genotype of the strains by pulsed-field gel electrophoresis (PFGE) and analyzed some phenotypic characteristics, such as O serotype, enzyme and mucous production, antibiotics susceptibility, and motility. PFGE allowed the typification of 97.1% of strains, revealing the presence of nine different genomic patterns. The pattern indicated as B was the most frequent, whereas patterns H and I were the most uncommon. Serotyping failed to identify 37.8% of strains and 29 out of 55 patients harbored almost one non-typable (NT) strain. During the five years of the study, we observed a progressive reduction of O6 and O10 types, but an increase of the O1 type and of NT strains. Most strains produced protease, hemolysin, and gelatinase, and were mobile. Several patients harbored the same serotype or genotype in sequential isolates, though characterized by a different susceptibility to antimicrobials. We did not observe a relationship between bacterial genotype and phenotype. This could be due to the fact that PFGE is not sensitive enough to detect subtle genotypic differences. The epidemiological importance of the genotypic characterization of bacteria-colonizing CF subjects and the surveillance measures to be adopted in CF centers are briefly discussed.     

Pyocin typing of Pseudomonas aeruginosa isolates from children with cystic fibrosis.

Pyocin typing and serotyping of 433 strains of Pseudomonas aeruginosa from children with cystic fibrosis (CF) showed that pyocin type 9 was predominant, particularly in association with polyagglutinating serotype. The common pyocin groups, 1, 5 and 10, made up only 20% of these isolates in contrast to reported rates of up to 89% in other studies using non-CF strains. No strains of pyocin type 3 were found. Polyagglutinating strains made up 72% of strains from patients colonized with P. aeruginosa for more than 12 mths. Pyocin type 9 was associated with 93% of polyagglutinating strains. The parallel between pyocin type 9 and polyagglutinating serotype suggests that these may both be characteristics acquired by P. aeruginosa colonizing patients with CF. Because of confounding between duration of colonization and exposure to cross-infection, this study does not allow definition of the role of cross-infection in determining the characteristics of these strains in most patients. In siblings, however, evidence supports a role for cross-infection either between siblings or from a common source. In 6 pairs of siblings studied, each pair had at least 1 pyocin group in common concurrently, either at entry to the study or after an interval of several months. Identical and unusual pyocin groups were recognized in samples obtained on the same day from pairs of siblings. More studies are needed to compare results of pyocin typing with methods such as genome fingerprinting to characterize these strains and determine whether the observed distribution of pyocin groups in CF isolates is related to cross-infection or whether the combination of pyocin type 9 with polyagglutinating serotype is a characteristic of CF strains.     

Auxotrophic variants of Pseudomonas aeruginosa are selected from prototrophic wild-type strains in respiratory infections in patients with cystic fibrosis.

Twenty-four nutritionally dependent (auxotrophic) Pseudomonas aeruginosa strains were isolated from 20 cystic fibrosis (CF) patients and tested for their amino acid requirements. Two different methods were necessary to identify the nutritional status of all isolates. Methionine was the most common single amino acid required (9 of 24 isolates), followed by leucine and arginine or ornithine. In total, a requirement for 12 different compounds or combination of compounds was demonstrated. Auxotrophic and prototrophic pairs of isolates from the same patient were compared by macrorestriction analysis of DNA in pulsed-field gel electrophoresis. Thirteen of 18 pairs analyzed presented identical restriction fragment length polymorphism profiles following digestion of DNA with XbaI. Three of the remaining pairs showed percentage similarities of 77, 91, and 98%, and the profiles of two pairs could not be compared because of the excessive degradation of their DNA. These results suggest that auxotrophic and prototrophic P. aeruginosa isolates colonizing the same CF patient constitute an isogenic group and raise the possibility that auxotrophs are selected from the prototrophic population during the course of pulmonary infection in CF patients.     

Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients

In total, 40 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients were included in this study. Twenty of these were collected in 1994 and 1997, from six CF patients, and the rest were collected from different CF patients in 2000 and 2001. The relative expression of mRNA for the efflux pump protein MexY was determined by real-time PCR and correlated with susceptibilities to amikacin and tobramycin. The chromosomal genes mexZ , rplY , galU , PA5471 and nuoG , which were found to have a role in the gradual increase in MICs of aminoglycoside antibiotics in laboratory mutants of P. aeruginosa , were analysed. MexY mRNA overproduction was found in 17/20 isolates collected in 1994 and 1997, and was correlated with decreased susceptibility to aminoglycosides. Alteration of the MexXY–OprM efflux system has been the main mechanism of resistance to aminoglycoside antibiotics in CF P. aeruginosa isolates over the 3-year period. In several isolates, expression of the PA5471 gene product might have some effect on elevated MICs of aminoglycosides. Inactivation of rplY , galU and/or nuoG may explain the gradual increase in MICs of aminoglycosides in laboratory mutants but probably not in the CF environment, as rplY and galU were unaltered in all isolates, and nuoG was not expressed in only one isolate. No 16S rRNA A-site mutations were found in any of the four copies of the gene in 13 investigated isolates.     

Cross-reactivity of Pseudomonas aeruginosa antipilin monoclonal antibodies with heterogeneous strains of P. aeruginosa and Pseudomonas cepacia.

Much of the morbidity and mortality in patients with cystic fibrosis (CF) is secondary to pulmonary infections with Pseudomonas aeruginosa and, more recently, with Pseudomonas cepacia. Prevention of colonization and subsequent infection would be a useful therapeutic strategy. The pili (fimbriae) of P. aeruginosa are a potential vaccine antigen, as they have been implicated in binding to respiratory epithelium and appear to have limited antigenic diversity. Monoclonal antibodies (MAbs) raised to P. aeruginosa pilin demonstrated significant cross-reactivity, as four of five P. aeruginosa strains with known pilin sequences and 10 of 15 P. aeruginosa clinical isolates hybridized by immunoblot with at least one of the three MAbs tested. The P. cepacia strains demonstrated minimal cross-reactivity with these MAbs, as only 2 of 16 strains hybridized immunologically. The three MAbs decreased the adherence of 35S-labeled P. aeruginosa PA1244 to bovine tracheal cells by 56, 45, and 31%. One of these MAbs decreased the adherence of strains P. aeruginosa PAO1 and P. cepacia 249 to CF epithelial cells by 46 and 25%, respectively. While antibodies to Pseudomonas pili must be shown to be protective in patients with CF, these studies give support for a multivalent vaccine strategy using P. aeruginosa pilin as the immunogen.