Evaluation of a nine-locus variable-number tandem-repeat scheme for typing of Pseudomonas aeruginosa.

Repeat numbers at nine variable-number tandem-repeat (VNTR) loci were determined for 177 isolates of Pseudomonas aeruginosa representing 77 strains distinguished by pulsed-field gel electrophoresis (PFGE). Eight loci provided for discrimination similar to that provided by PFGE, with variation at the ninth locus (ms61) sometimes allowing discrimination within a PFGE-defined type. The Liverpool and Midlands 1 strains, which are common among patients with cystic fibrosis in the UK, could be unambiguously identified by their characteristic VNTR profiles. In rare cases, the repeat number at the ninth locus alone provided discrimination among isolates that were distinct according to PFGE. In each case, the two isolates shared the same bla(OXA-50-like) allele and belonged to the same oprD sequence type group, supporting the VNTR results in suggesting that they are similar.

Transmission of Pseudomonas aeruginosa in children with cystic fibrosis attending summer camps in The Netherlands.

BACKGROUND: Cross-infection of Pseudomonas aeruginosa has been reported to occur at holiday camps for children with Cystic Fibrosis (CF) with varying frequency. The study aimed to establish the degree of transmission resulting in subsequent infection of P. aeruginosa among CF children (n=80) attending holiday camps in The Netherlands. METHODS: The study was performed in the summer of 2001 in four camps organised simultaneously at different locations. Sputum was collected on day 1 of the holiday, and three and six months later. Different morphotypes of P. aeruginosa from sputum were genotyped by AFLP analysis. Criteria were defined for the degree of evidence of transmission. RESULTS: There were 18 cases possible, 2 cases of probable transmission and 1 case of highly probable transmission. Two predominant types of P. aeruginosa were found (types 18 and 23). Type 18 was already prevalent on day 1 mostly in younger children and was involved in eleven cases of transmission; type 23 was involved in six cases of transmission among older children. CONCLUSIONS: There was a considerable risk of transmission of P. aeruginosa during holiday camps for CF children in The Netherlands. Two genotypes of P. aeruginosa appeared to be easily transmissible, one of which seemed common in the Dutch CF population.

Characterisation of Exiguobacterium aurantiacum isolates from blood cultures of six patients.

Exiguobacterium spp. are alkaliphilic, halotolerant, non-spore-forming Gram-positive bacilli, hitherto uncharacterized from human infections. Six isolates of Exiguobacterium aurantiacum were obtained from patients with bacteraemia, three of whom had myeloma. All isolates formed orange-yellow pigmented colonies on blood agar, were catalase- and DNase-positive, and grew on nutrient agar at pH 10 and in the presence of NaCl 6% w/v. The six isolates were susceptible to all antimicrobial agents tested and were uniform in their fatty acid and mass spectrum profiles.

European-wide distribution of Pseudomonas aeruginosa clone C.

A collection of 77 epidemiologically unrelated Pseudomonas aeruginosa isolates was screened for the occurrence of clone C isolates by the appearance of characteristic SpeI fragment patterns obtained by pulsed-field gel electrophoresis. Three strains with a clone C characteristic SpeI fragment pattern were found which also harbored the clone C-specific plasmid either in the free form or chromosomally integrated. Genomic islands were detected in the new clone C strains, as in already characterized clone C strains. Clone C not only infected cystic fibrosis patients throughout Europe, but was also found in the UK as an isolate in urinary tract infections and in peritoneal dialysis fluid, in addition to an otitis media isolate. Therefore, P. aeruginosa clone C is widely distributed in Europe, with a broad pathogenic potential.

Survey of resistance of Pseudomonas aeruginosa from UK patients with cystic fibrosis to six commonly prescribed antimicrobial agents.

BACKGROUND: Respiratory infection with Pseudomonas aeruginosa is very common in patients with cystic fibrosis (CF) but antimicrobial resistance rates of CF isolates across the UK are largely unknown. METHODS: The susceptibility of 417 CF patient isolates of P aeruginosa from 17 hospitals to six commonly prescribed antibiotics were examined. Isolates were tested by an agar break point dilution method and E-tests according to British Society of Antimicrobial Chemotherapy guidelines. Genotyping of isolates was performed by XbaI DNA macrorestriction and pulsed field gel electrophoresis. RESULTS: 38% of isolates were susceptible to all of the agents tested; almost half were resistant to gentamicin compared with ceftazidime (39%), piperacillin (32%), ciprofloxacin (30%), tobramycin (10%), and colistin (3%). Approximately 40% were resistant to two or more compounds with ceftazidime in combination with gentamicin, piperacillin or ciprofloxacin being the most common cross resistances. Resistance rates were generally similar to those reported recently from the USA and Germany. A selection of resistant isolates proved to be predominantly genotypically distinct by XbaI DNA macrorestriction but six pairs from three centres had similar genotypes. CONCLUSIONS: The level of resistance to front line antipseudomonal agents, with the exception of colistin, is disturbingly high. The prudent use of antimicrobial drugs and closer monitoring of accumulation of resistant strain populations should be actively considered.

Increased sputum amino acid concentrations and auxotrophy of Pseudomonas aeruginosa in severe cystic fibrosis lung disease.

BACKGROUND: Pseudomonas aeruginosa may undergo a phenotypic change from the wild (prototrophic) type to an auxotrophic phenotype in the course of respiratory infection in patients with cystic fibrosis. The clinical significance of this is unclear. A study was undertaken to investigate whether the presence of auxotrophs of P aeruginosa in the sputum of patients with cystic fibrosis correlated with severity of respiratory disease, and whether increased sputum concentrations of amino acids were associated with the emergence of these forms. METHODS: Sixty adult patients with cystic fibrosis, colonised by P aeruginosa, were recruited and baseline clinical data including lung function were recorded. Serial sputum samples were obtained before, during, and after infective exacerbations where possible. These samples were used for routine microbiological culture, assessment of auxotrophy of P aeruginosa, measurement of amino acid content, and neutrophil elastase assay. RESULTS: Auxotrophy was common in patients with cystic fibrosis and 20 (33%) had a mean percentage auxotroph count of more than 50% total cfu/ml. The mean percentage auxotroph count was inversely correlated with forced expiratory volume in one second (FEV(1); tau = -0.194, p = 0.031). The median sputum amino acid concentration of the group was 12.5 mmol/l (range 0.13-40.6). The mean amino acid concentration in 33 subjects during infective exacerbations was 18.2 mmol/l (95% CI 15.1 to 21.3) compared with 12.3 mmol/l (95% CI 9.8 to 14.8) when well (p = 0.001). The amino acid content of sputum was inversely correlated with FEV(1) (tau = -0.253, p = 0.005). CONCLUSIONS: P aeruginosa frequently exhibits auxotrophy in patients with cystic fibrosis, particularly in those with severe underlying pulmonary disease. The sputum amino acid content of patients with cystic fibrosis is high during infective exacerbations and correlates with pulmonary disease severity.

Distribution of genes encoding putative transmissibility factors among epidemic and nonepidemic strains of Burkholderia cepacia from cystic fibrosis patients in the United Kingdom.

In the last 15 years, Burkholderia cepacia has emerged as a significant pathogen in cystic fibrosis (CF) patients, mainly due to the severity of infection observed in a subset of patients and the fear of transmission of the organism to noncolonized patients. Although patients who deteriorate rapidly cannot be predicted by microbiological characteristics, three genetic markers have been described for strains that spread between patients. These are the cblA gene, encoding giant cable pili; a hybrid of two insertion sequences, IS1356 and IS402; and a 1.4-kb open reading frame known as the B. cepacia epidemic strain marker (BCESM). The latter two are of unknown function. An epidemic strain lineage was previously identified among CF patients in the United Kingdom that apparently had spread from North America and that was characterized by a specific random amplified polymorphic DNA (RAPD) pattern. We searched for the described genetic markers using specific PCR assays with 117 patient isolates of B. cepacia from 40 United Kingdom hospitals. Isolates were grouped according to genomovar and epidemic strain lineage RAPD pattern with a 10-base primer, P272. A total of 41 isolates from patients in 12 hospitals were classified as the epidemic strain, and 40 of these were distributed in genomovars IIIa (11 isolates), IIIb (1 isolate), and IIIc (28 isolates). All isolates of the epidemic strain were positive for the cblA gene and BCESM, but two lacked the insertion sequence hybrid. None of the 76 sporadic isolates contained cblA or the insertion sequence hybrid, but 11 of them were positive for BCESM. Nonepidemic isolates were distributed among genomovars I or IV (9), II (49), IIIa (11), IIIb (3), and IIIc (4). There were three clusters of cross-infection (one involving two patients and two involving three patients) with isolates of genomovar II. We conclude that in the United Kingdom, a single clonal lineage has spread between and within some hospitals providing care for CF patients. The presence of the cblA gene is the most specific marker for the epidemic strain. We recommend that all isolates of B. cepacia from CF patients should be screened by PCR to influence segregation and infection control strategies.

Evaluation of three oligonucleotide primer sets in PCR for the identification of Burkholderia cepacia and their differentiation from Burkholderia gladioli.

AIMS: To evaluate three oligonucleotide primer pairs--two specific for 16S and 23S rRNA sequences of Burkholderia cepacia, and the third specific for internal transcribed spacer region of 16S-23S sequences of B gladioli--for the identification and differentiation of reference and clinical strains of these and other species. METHODS: The three primers sets were applied in polymerase chain reaction (PCR) to a collection of 177 clinical isolates submitted for identification from diagnostic laboratories as presumed B cepacia. RESULTS: At an annealing temperature of 63 degrees C, all eight B cepacia and four B gladioli reference strains reacted with their specific primers. B vandii was the only other species that was positive with both B cepacia primers but five Burkholderia or Ralstonia species reacted with one of these primers. Seventy eight isolates were typical of B cepacia in biochemical tests and 75 of these reacted with specific primers; three, however, were positive with the B gladioli primers. Fifteen asaccharolytic isolates were confirmed as B cepacia by PCR but other non-fermenting Gram negative species were negative with each of the primers. CONCLUSIONS: PCR using 16S rRNA sequences is recommended for identification of B cepacia that give atypical results in biochemical tests.

Regional microbiology of the cystic fibrosis lung: a post-mortem study in adults.

Although the majority of patients with cystic fibrosis (CF) become chronically colonized with Pseudomonas aeruginosa, the mode of acquisition of infection remains unclear. Epidemiological studies using genotyping techniques suggest that person-to-person transmission of this organism may occur. All these studies have utilized sputum or throat swab samples. We have studied the regional microbiological flora of the lungs of five CF patients at post-mortem and genotyped P. aeruginosa strains found therein and in the major airway. We have shown that although in most cases major airway secretions accurately reflect the peripheral lung flora, in cases of multiple strain carriage, major airway cultures may not reflect all strains present in the periphery of the lung. This finding has implications for the interpretation of epidemiological studies that use genotyping of strains from sputum and throat swab samples to assess possible routes of transmission.

Persistence of Pseudomonas aeruginosa strains in respiratory infection in AIDS patients.

OBJECTIVES: To establish the clinical pattern of Pseudomonas aeruginosa respiratory infections in HIV-seropositive patients and to determine whether repeated isolation of the organism represents reinfection or recurrence and to assess whether common source, nosocomial infection occurred. DESIGN AND METHODS: Evaluation of the clinical pattern of P. aeruginosa respiratory infections by case note review and epidemiological characterization of P. aeruginosa by serotype determination and Xbal DNA macrorestriction analysis. Serum sensitivity testing of strains was performed to further define phenotypic characteristics of the isolated organisms. RESULTS: Seventy-three per cent (29 out of 40) of individuals had P. aeruginosa isolated on two or more occasions in the setting of clinical respiratory infection. Overall, 85% had evidence of P. aeruginosa to within 2 months of study completion or death. Epidemiological characterization revealed persistence of unique single strains in 93% of individuals where multiple isolates were available for testing, whereas only two patients harboured a common strain. The serotype distribution of strains was similar to that reported from non-HIV-positive patients. CONCLUSIONS: Once established, eradication of P. aeruginosa from the respiratory tract of HIV-seropositive individuals with advanced immunosuppression is problematic and a chronic infective state appears common. There was no evidence of nosocomial transmission. Serotype loss and development of sensitivity to normal human serum were both observed and were highly correlated. This represents truncation of O-antigenic lipopolysaccharide on the cell surface of P. aeruginosa and may reflect progression to phenotypes commonly associated with chronic infection in other clinical settings such as cystic fibrosis.

Complementation of methionine auxotrophs of Pseudomonas aeruginosa from cystic fibrosis.

Auxotrophic Pseudomonas aeruginosa are exclusive to respiratory infections in cystic fibrosis (CF) and bronchiectatic patients, and isolates require specific amino acids for growth on minimal media, particularly methionine. Since auxotrophic and prototrophic P. aeruginosa from CF are identical by genotyping, we investigated the genetic events leading to methionine auxotrophy (Met-). Most (10/13) Met- strains had the same pattern of growth on methionine precursors and required methionine exclusively for growth. Back mutation to prototrophy was very low (frequencies 10(-8) to <10(-10)). Complementation of the mutations leading to auxotrophy was achieved for five strains with a genomic library of P. aeruginosa PAO1. Strains with different patterns of growth on methionine precursors were complemented by clones with different restriction patterns, while identical clones complemented strains with the same pattern of growth on methionine precursors. Methionine auxotrophy in P. aeruginosa from CF results from stable chromosomal mutations, and the commonest defect is probably in gene(s) encoding enzymes that convert homocysteine to methionine.

The high amino-acid content of sputum from cystic fibrosis patients promotes growth of auxotrophic Pseudomonas aeruginosa.

Many isolates of Pseudomonas aeruginosa from cystic fibrosis (CF) patients are auxotrophic and require amino acids for growth. A quantitative assay was used to determine the total content of free amino acids of sputum sol-phase extracts from CF and non-CF patients to assess the presence of amino acids in the airway. CF patients colonised with auxotrophic P. aeruginosa had a higher sputum amino-acid content (mean 6.77 mg/ml) than those colonised with prototrophs (mean 3.77 mg/ml); overall, CF specimens (mean 5.70 mg/ml) had a higher amino-acid content than non-CF samples (2.52 mg/ml). The amino-acid profile of sputum extracts was assessed by one-dimensional thin layer chromatography (TLC). Several amino acids were identified in the extracts, in particular, leucine, isoleucine, phenylalanine, tyrosine, alanine, serine and methionine or valine or both. All sputum specimens except two (which contained < 1.5 mg of amino acids/ml), promoted the growth, of 34 auxotrophic strains of P. aeruginosa from CF patients in a minimal medium. These results indicate, therefore, that amino acids are plentiful in the sputum of CF patients and are able to supply the requirements of auxotrophic strains. It is suggested that the increased amino-acid content in the airways of CF patients plays a significant role in the selection and maintenance of nutritionally deficient P. aeruginosa.

Home-use nebulizers: a potential primary source of Burkholderia cepacia and other colistin-resistant, gram-negative bacteria in patients with cystic fibrosis.

Inhalation of aerosols contaminated with gram-negative bacteria generated from home-use nebulizers used by cystic fibrosis (CF) patients may be a primary route for bacterial colonization of the lung. Burkholderia cepacia was isolated from 3 of [corrected] 35 home-use nebulizers, and Stenotrophomonas maltophilia was isolated from 4 of 35 home-use nebulizers. Sputum cultures for two patients whose nebulizers were contaminated with B. cepacia did not yield the organism. However, DNA macrorestriction analysis by pulsed-field gel electrophoresis confirmed that one of two strains of B. cepacia recovered from the nebulizer of a third patient was also present in the sputum of that patient. Although Pseudomonas aeruginosa was isolated from 34 patients, none of the nebulizers were positive for the organism. Sixty-nine percent of nebulizers were contaminated, and up to 16 different environmental colistin-resistant, gram-negative species were identified. The heaviest contamination was found beneath the chamber atomizer. A questionnaire survey showed that the majority of patients (28 of 34) were receiving nebulized colistin and/or gentamicin. Patients who followed recommended instructions for good nebulizer hygienic practice and paid particular attention to drying had minimal or no contamination of their nebulizers.

Type characterisation and antibiotic susceptibility of Burkholderia (Pseudomonas) cepacia isolates from patients with cystic fibrosis in the United Kingdom and the Republic of Ireland.

The spread of Burkholderia cepacia among cystic fibrosis (CF) patients in the UK prompted an investigation into whether an epidemic strain was responsible. A total of 366 B. cepacia isolates from 178 CF patients in 17 centres was examined by ribotyping and pulsed-field gel electrophoresis (PFGE). Associations were also sought between antibiotic resistance and strain type. More than 50 ribotype patterns were found but one, termed ribotype 1, was identified from 68 patients in eight centres. One centre had a single patient with this type while, in others, most or all patients harboured this organism. Small clusters of apparent cross-colonisation within centres were also evident for some other ribotypes. PFGE confirmed that ribotype 1 isolates were genetically similar. Ribotype 1 isolates were not markedly more resistant to antimicrobial agents than were other isolates, and the MICs of individual antibiotics were no more tightly clustered for ribotype 1 isolates than for others. Most isolates were resistant to ciprofloxacin, amikacin, gentamicin, tobramycin, carbenicillin, cefuroxime, cefotaxime, imipenem, biapenem, chloramphenicol, tetracycline, trimethoprim and sulphamethoxazole, but > or = 77% were susceptible to ceftazidime, piperacillin, piperacillin/ tazobactam and meropenem. We conclude that numerous strains of B. cepacia colonise CF patients in the UK and Ireland but that one epidemic strain has spread in at least eight centres. Isolates of this strain appear homogenous in total genomic profile but very variable in antibiotic susceptibility.

Auxotrophy of Burkholderia (Pseudomonas) cepacia from cystic fibrosis patients.

The nutritional status of 89 isolates of Burkholderia cepacia from 81 cystic fibrosis (CF) patients was evaluated. Forty of the isolates, from 38 patients, were not able to grow in a minimal medium containing glucose and mineral salts only and were thus auxotrophs. In contrast, all of 29 isolates from non-CF (clinical and environmental) sources were prototrophic. Addition of a pool of amino acids to the minimal medium was sufficient to promote growth of all tested CF auxotrophic isolates. Indeed, phenylalanine, tyrosine, cysteine, methionine, and histidine alone or in combination were required for growth by the majority of the nutritionally deficient B. cepacia isolates. Furthermore, extracts of sputum from CF patients, when added to minimal medium, promoted growth of 29 auxotrophic B. cepacia isolates regardless of their amino acid requirements. Finally, auxotrophic and prototrophic isolates from the same patient exhibited a conserved genotype, as determined by macrorestriction analysis of chromosomal DNA. These results suggest that the auxotrophic mutants are selected from the prototrophic population and maintained by the nutritionally rich environment of the CF airways.

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.

Colonisation of lung allografts with Pseudomonas aeruginosa in heart-lung transplant recipients with cystic fibrosis.

Six patients (four with cystic fibrosis, two with bronchiectasis) harboured Pseudomonas aeruginosa in the lung before heart-lung transplantation. Three of the patients with cystic fibrosis were colonised by strains of different genotype postoperatively, and the colonisation tended to be short lived.

Epidemic of Pseudomonas cepacia in an adult cystic fibrosis unit: evidence of person-to-person transmission.

An epidemic of Pseudomonas cepacia occurred in an adult cystic fibrosis center in the United Kingdom, despite a policy of segregation of infected and noninfected patients within the hospital. Investigation of the outbreak by ribotyping and pulsed-field gel electrophoresis to characterize P. cepacia strain genomes together with inquiry into social contacts between patients revealed evidence of person-to-person transmission outside the hospital environment. Segregation policies aimed at reducing the spread of this infection in the cystic fibrosis community need to encompass patient contacts outside the hospital environment.