Direct sputum analysis of Pseudomonas aeruginosa macrorestriction fragment genotypes in patients with cystic fibrosis

The distribution of bacterial populations in the airways of 13 patients with cystic fibrosis who were colonized for 6 – 23 years with Pseudomonas aeruginosa was investigated by genotyping of bacterial chromosomes directly isolated from 21 sputa. After removal of host material from sputum by hypotonic cell lysis and repetitive washing and centrifugation steps, agarose-embedded bacterial cells were lysed, residual eukaryotic DNA separated by field inversion gel electrophoresis, and the purified bacterial chromosomes subjected to macrorestriction fragment pattern and Southern analyses. Bacterial populations consisted of a single P. aeruginosa clone in 17 sputa, of which more than one clonal variant was apparent in two SpeI fragment fingerprints. Two clones of P. aeruginosa and another species co-existed in four samples. Genomically homogeneous populations of P. aeruginosa are characteristic for chronically colonized lungs in most cases of cystic fibrosis. Received: 25 April 1997     

Molecular epidemiology of Staphylococcus aureus strains colonizing the lungs of related and unrelated cystic fibrosis patients

Background: Cystic fibrosis (CF) patients can become persistently colonized with Staphylococcus aureus. This is initiated at an early age and may continue until or sometimes even during adolescence. Little is known about the epidemiology and cross-infectivity of S. aureus in CF patients, whether via the environment or person to person.
Method: S. aureus isolates ( n = 189) from six unrelated CF aptients and six pairs of CF siblings were genetically typed by arbitrary primed polymerase chain reaction (AP-PCR) assays.
Results: This longitudinal study revealed 35 different genotypes among the 189 isolates; the median number of types in a patient was three (range 1–6). One common S. aureus genotype was found in six patients and involved 20% of all isolates analyzed. Ultimately, in most of the patients long-term colonization with a single genotype was observed. In several, but certainly not all, pairs the siblings became persistently colonized with isolates that could not be discriminated by the typing method used; different S. aureus genotypes were isolated on an incidental but relatively frequent basis. Only one pair of siblings never shared identical isolates at any time during the screening period.
Conclusions: In five of six cases, identical isolates were shared by CF siblings at a certain time. This suggests intra-family transmission or the presence of a common environmental source. The fact that in most of the CF sibling pairs different genotypes of S. aureus caused the ultimate long-term colonization indicates that, despite regular cross-colonization, patient characteristics select the S. aureus strain best adapted to the affected lung. Some genotypes may be particularly prevalent in the CF patient population, but additional studies are needed to confirm this.     

Epidemiologic characterization of Pseudomonas aeruginosa in patients with cystic fibrosis

Objective   To determine persistence and variability of colonization with Pseudomonas aeruginosa in cystic fibrosis patients over long time periods, and to look for possible cross-colonization.
Methods   In total, 469 Pseudomonas aeruginosa isolates were obtained from 30 patients during the period from April 1994 to April 1996. The sources were mainly sputum and a few deep throat swabs. All grown strains dissimilar in macromorphology were processed separately. Typing with PFGE was carried out by contour-clamped homogeneous electric field electrophoresis. Genomic DNA was subjected to the rare-cutting restriction enzyme Spe I. For pyocin typing, the procedure described by Fyfe was applied.
Results   After typing with PFGE, we observed 40 restriction profiles. Eighteen different pyocin types were found. The most frequent pyocin type was type 3, followed by types 1 and 5. Twenty-two patients were persistently colonized by one clone specific and different for each patient, and four were co-colonized by a second clone also different for each of these patients. Cross-colonization had apparently been rare in the cystic fibrosis center of Leipzig.
Conclusions   Typing with PFGE is well suited for detailed investigations of colonization with Pseudomonas aeruginosa in cystic fibrosis patients. Pyocin typing can provide additional information for epidemiologic purposes.     

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.     

First report of cystic fibrosis mutations in Libyan cystic fibrosis patients

Background: There are few data on the molecular basis of Cystic Fibrosis (CF) in North Africa, probably due to under-diagnosis.

Aim: This is the first study of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in the Libyan population.

Subjects and methods: This study analysed the complete coding region and flanking intronic sequences of the CFTR gene in 10 unrelated Libyan CF patients.

Results: This study identified four mutations (F508del, c.1670delC, N1303K and E1104X), with a high frequency of the latter.

Conclusion: Identification of CF mutations facilitates molecular investigation of cystic fibrosis in the Libyan population and helps to provide effective genetic counselling among CF families.     

The relationship of eosinophil granule proteins to ions in the sputum of patients with cystic fibrosis

Increased sputum levels of eosinophil granule proteins have been reported despite normal eosinophil numbers in peripheral blood and in the lung in cystic fibrosis (CF). Mechanisms of eosinophil priming and activation are still unclear in CF. In the present study we investigated whether ion concentrations in the sputa of CF patients are related to eosinophil activity. We assessed concentrations of eosinophil cationic protein (ECP), eosinophil protein X (EPX), major basic protein (MBP) and ions (Na+, Cl-, Ca2+, Mg2+) in sputum samples of 29 children with CF as well as in 10 controls with bronchial asthma. Patients with CF demonstrated significantly higher levels of ECP, Na+, Cl- and Ca2+ levels than asthmatics (P < 0.04, P < 0.0001, P < 0.0001, P < 0.02). No differences were seen between concentrations of EPX and Mg2+ in the two groups. In CF, eosinophil granule proteins correlated significantly with Ca2+ and Mg2+ concentrations (ECP, P < 0.0001, r = 0.65, P < 0.0001, r = 0.66; MBP, P < 0.03, r = 0.41, P < 0.03, r = 0.42), furthermore inversely with Cl- concentrations (ECP, P < 0. 0003, r = - 0.63; EPX, P < 0.02, r = - 0.45; MBP, P < 0.03, r = - 0. 41) but not with Na+ levels. ECP, Na+ and Cl- were also correlated with lung function variables (FVC, P < 0.04, r = - 0.38, P < 0.02, r = 0.44, P < 0.03, r = 0.41; FEV1, P < 0.007, r = - 0.49, P < 0.006, r = 0.5, P < 0.008, r = 0.48; MEF50, P < 0.003, r = - 0.54, NS, P < 0.03, r = 0.42; MEF25, P < 0.039, r = - 0.4, P < 0.005, r = 0.51, P < 0.05, r = 0.37). Our results demonstrated a significant relationship of eosinophil degranulation and ions in CF, indicating that ion composition in CF sputa may be at least partly be responsible for high levels of eosinophil products despite low eosinophil numbers.     

Chloride transport in cultured nasal epithelium of cystic fibrosis patients

In this study, nasal polyp epithelial cells from control and cystic fubrosis (CF) patients were cultured using a method which allows multiple passages. The cells were tested in Ussing chamber experiments to study transcellular ion transport. Cultured CF nasal polyp cells did not exhibit spontaneous transcellular chloride transport in the presence of amiloride, in contrast to normal cells. Forskolin increased the short circuit current (I _sc) in control but not CF cells. Forskolin and isoproterenol increased the cAMP levels in control and CF cells. Histamine, bradykinin and isoproterenol transiently increased the intracellular calcium level and caused a parallel increase of the transcellular chloride current in both normal and CF cells. The transient effects of isoproterenol were not sensitive to the beta blocker atenol and could not be mimicked by forskolin. We conclude that in cultured nasal polyp cells a difference in chloride transport activity between CF and control cells is retained following multiple passages. Our results suggest that the active state of chloride channels in nasal polyp cells does not require activation of a second messenger pathway. This apparently spontaneous activity appears to be reduced in CF cells. The calcium- but not the cAMP-dependent activation of transepithelial chloride secretion is at least partially preserved in cultured CF airway epithelium.     

Bacteria of the genus Dyella can chronically colonise the airways of patients with cystic fibrosis and elicit a pronounced antibody response

A patient with cystic fibrosis became chronically colonised with an unusual non-fermenting Gram-negative rod that could be cultured on Burkholderia cepacia selective agar. Phenotypic characterisation by VITEK-2 suggested identification as Elizabethkingia meningoseptica, however 16S rRNA gene sequencing revealed it belonged to a putative novel species of genus Dyella. Thirty months after the initial detection, the patient produced a high level of precipitating antibodies against the bacterium.     

Factors associated with mucoid transition of Pseudomonas aeruginosa in cystic fibrosis patients

Although the mucoid form of Pseudomonas aeruginosa (Pa) is largely responsible for the progression of lung disease in cystic fibrosis (CF), the relationship between factors relating daily-care regimes to mucoidy acquisition are as yet poorly investigated. Fifty-two CF patients registered at the CF centre of Dijon, France, were retrospectively evaluated from the date of Pa colonization either to the first -positive sputum culture for mucoid Pa (n = 26) or to the last culture in which the Pa remained non-mucoid (n = 26). All clinical, pathological and therapeutic events were recorded. The association between the parameters collected and mucoid transition of Pa was assessed in a Cox model with time-dependant covariables. The mean follow-up was 4.7 ± 4.3 years. Three independent parameters were associated with the higher risk of mucoid transition of Pa: persistence of Pa in sputum (OR 7.89; p <0.01), use of inhaled bronchodilators (OR 3.40; p = 0.04), and the use of inhaled colimycin (OR 4.04; p = 0.02). Isolation of Staphylococcus aureus, Haemophilus influenzae or Streptococcus pneumoniae in sputum was associated with a lower risk (OR 0.24; p < 0.01). Mucoid transition of Pa was associated with variables that reflected the severity of both lung disease and Pa colonization. Although they do not lead to prophylactic measures, these results corroborate the need to avoid Pa persistence.     

Typing of Pseudomonas aeruginosa strains in Norwegian cystic fibrosis patients

Objectives   Typing of Pseudomonas aeruginosa isolates from Norwegian cystic fibrosis (CF) patients with chronic Pseudomonas lung infection in order to see whether cross-infection might have occurred.
Methods   Isolates from 60 patients were collected during the years 1994–98, and typed by pulsed field gel electrophoresis.
Results   Seventy-one strains were identified. One large cluster of identical strains included 27 patients, and 13 smaller clusters of 2–4 patients were found (26 patients). Seven patients had a strain not shared by other patients (private strains). Harboring the main cluster strain was significantly associated with participation in summer camps and training courses ( P  = 0.004, chi-squared test). There were no associations with regular admissions to hospital (intravenous antibiotic courses) or smaller social gatherings of short duration. Small clusters and private strains were not associated with any of the risk factors. All strains were sensitive to colistin. The minimal inhibitory concentrations were generally lower in Norwegian P. aeruginosa strains compared with isolates from Danish patients.
Conclusions   Our results indicate that cross-infection with P. aeruginosa between cystic fibrosis patients has occurred.     

Marker genes for the metabolic adaptation of Pseudomonas aeruginosa to the hypoxic cystic fibrosis lung environment

Pseudomonas aeruginosa is the leading pathogen of chronic cystic fibrosis (CF) lung infection. Life-long persistence in the inflamed and ever fluctuating CF lungs results in the selection of a variety of changes in P. aeruginosa physiology. Accumulating evidence suggests that especially metabolic changes support the survival and growth of P. aeruginosa within the hypoxic and nutritious CF mucus. To investigate if metabolic adaptations we described for hypermutable P. aeruginosa from late CF lung disease (Hoboth et al., 2009. J. Infect. Dis., pp. 118–130) may represent specific changes in response to the selective conditions within the oxygen-restricted CF mucus, we determined the expression of a set of genes during aerobic and hypoxic growth in LB and the artificial sputum medium ASM. We further focused on the regulation of the two isocitrate dehydrogenases Icd and Idh. Interestingly, both isoenzymes may replace each other under aerobic and hypoxic conditions. The NADPH- and RpoS-dependent Icd seems to be the leading isoenzyme under prolonged oxygen limitation and stationary growth phase. LacZ reporter analysis revealed that oxygen-restriction increased the expression levels of azu, cbb3-1, cbb3-2, ccpR, icd, idh and oprF gene, whereas himD and nuoA are increasingly expressed only during hypoxic growth in ASM. Overexpression of the anaerobic regulator Anr improved the expression of azu, ccpR, cbb3-2 and icd. In summary, expression of azu, cbb3-1, cbb3-2, ccpR, icd, idh, oprF, himD, and nuoA appeared to be beneficial for the growth of P. aeruginosa under hypoxic conditions indicating these genes may represent marker genes for the metabolic adaptation to the CF lung environment.     

Intracellular retention of lysosomal enzymes in cystic fibrosis

Multiple leakage of lysosomal enzymes from cystic fibrosis fibroblasts into the extracellular space has been suggested to occur but was not observed in these experiments. Both extracellular and intracellular levels of enzyme activity were within the normal range. The release of lysosomal enzymes from individual cell lines was seen to be affected both by intrinsic properties of each cell line and by many external factors.     

Identification of Bordetella spp. in respiratory specimens from individuals with cystic fibrosis.

Bordetella spp. are not normally included when considering the opportunistic bacterial species that are typically involved in respiratory tract infections in individuals with cystic fibrosis (CF). By using a combination of bacterial genotyping and 16S rDNA sequencing, Bordetella spp. were identified in cultures obtained from 43 individuals with CF. Most (n = 23) patients were infected with Bordetella bronchiseptica/parapertussis; five were infected with Bordetella hinzii, four with Bordetella petrii, three with Bordetella avium, and eight with unidentified Bordetella spp. Consideration should be given to the presence of these organisms in the evaluation of CF sputum cultures.     

Oral bacterial community dynamics in paediatric patients with malignancies in relation to chemotherapy-related oral mucositis: a prospective study

The role of oral bacteria in the development of chemotherapy-related oral mucositis has not been fully elucidated. This study aimed to investigate oral bacterial community diversity and dynamics in paediatric patients with malignancies in relation to the occurrence of oral mucositis. Patients with malignancies (n = 37) and reference individuals without known systemic disorders (n = 38) were recruited. For patients, oral bacterial samples were taken from mucosal surfaces both at the time of malignancy diagnosis and during chemotherapy. If oral mucositis occurred, samples were taken from the surface of the mucositis lesions. Oral mucosal bacterial samples were also taken from reference individuals. All samples were assessed using a 16S ribosomal RNA gene 454 pyrosequencing method. A lower microbial diversity (p < 0.01) and a higher intersubject variability (p < 0.001) were found in patients as compared with reference individuals. At the time of malignancy diagnosis (i.e. before chemotherapy) patients that later developed mucositis showed a higher microbial diversity (p < 0.05) and a higher intersubject variability (p < 0.001) compared with those without mucositis. The change of bacterial composition during chemotherapy was more pronounced in patients who later developed mucositis than those without mucositis (p < 0.01). In conclusion, we found a higher microbial diversity at the time of malignancy diagnosis in patients who later develop oral mucositis and that these patients had a more significant modification of the bacterial community by chemotherapy before the occurrence of mucositis. These findings may possibly be of clinical importance in developing better strategies for personalized preventive management.     

Complete identification of cystic fibrosis transmembrane conductance regulator mutations in the CF population of Saguenay Lac-Saint-Jean (Quebec, Canada)

Over the past few years, we have conducted a systematic study of 230 cystic fibrosis (CF) chromosomes in the Saguenay Lac-Saint-Jean (SLSJ) population which has a high CF incidence (1/936 live births). We identified 11 mutations accounting for 100% of the CF chromosomes found in patients born in SLSJ. Our results indicate that denaturing gradient gel electrophoresis (DGGE) is a powerful method of identifying CF mutations. They have also considerable implications for genetic counselling and molecular characterization of doubtful patients. They make carrier screening technically feasible in this population.     

Different colonization patterns of Aspergillus terreus in patients with cystic fibrosis

Aspergillus terreus is a common soil saprophyte. After Aspergillus fumigatus and Scedosporium apiospermum it ranks third amongst the filamentous fungi colonizing the airways of patients with cystic fibrosis. In this context, the clinical presentation of A. terreus infection mainly corresponds to allergic broncho-pulmonary aspergillosis. In the work presented here, we studied colonization patterns of A. terreus in CF patients by genotyping using nine short tandem repeat markers. A total of 115 clinical isolates from respiratory secretions collected from five French CF patients were studied. The number of isolates varied from 15 to 39 per patient, and the duration of the follow-up period ranged from 2 months to 7.5 years. Seventeen genotypes were identified, corresponding to three distinct colonization patterns. The first colonization pattern consisted of a chronic colonization by one dominant genotype associated with few other genotypes found only incidentally. The second colonization pattern consisted of a prolonged colonization by two distinct genotypes detected simultaneously. The last pattern was characterized by multiple different genotypes that were present only transiently. These results demonstrate the importance of genotyping clinical isolates before making conclusions about chronic colonization of the airways in CF patients in the case of repeated isolation of the fungus.     

Prevalence of hypermutator isolates of Achromobacter spp. from cystic fibrosis patients

Bacteria colonising the lungs of cystic fibrosis (CF) patients encounter high selective pressures. Hypermutation facilitates adaptation to fluctuating environments, and hypermutator strains are frequently isolated from CF patients. We investigated the prevalence of hypermutator isolates of Achromobacter spp. among patients affiliated with the CF Centre in Aarhus, Denmark. By exposure to rifampicin, the mutation frequency was determined for 90 isolates of Achromobacter spp. cultured from 42 CF patients; 20 infections were categorised as chronic, 22 as intermittent. The genetic mechanisms of hypermutation were examined by comparing DNA repair gene sequences from hypermutator and normomutator isolates. Achromobacter spp. cultured from 11 patients were categorised as hypermutators, and this phenotype was exclusively associated with chronic infections. Isolates of the Danish epidemic strain (DES) of Achromobacter ruhlandii cultured from patients from both Danish CF centres showed elevated mutation frequencies. The hypermutator state of Achromobacter spp. was most commonly associated with nonsynonymous mutations in the DNA mismatch repair gene mutS; a single clone had developed a substitution in the S-adenosyl-L-methionine-dependent methyltransferase putatively involved in DNA repair mechanisms, but not previously linked to the hypermutator phenotype. Hypermutation is prevalent among clinical isolates of Achromobacter spp. and could be a key determinant for the extraordinary adaptation and persistence of DES.     

Prevalence and dynamics of Lactobacillus sp. in the lower respiratory tract of patients with cystic fibrosis

No prevalence or dynamics analysis of Lactobacilli in the lung of cystic fibrosis (CF) patients has yet been conducted. In order to use them as probiotics in the treatment of Pseudomonas aeruginosa infection, we describe their lung epidemiology.Over a period of 8 months, we analyzed 279 sputum samples from 124 CF patients classified according to their P. aeruginosa Leeds status of colonization. A total of 137 strains belonging to 11 species were isolated. The prevalence of carriage was 61%. No difference in species diversity or frequency was observed according to Leeds criteria. The next step will be to focus on the strain level.     

Prospective study on nontuberculous mycobacteria in patients with and without cystic fibrosis

Recent reports indicated an increase of nontuberculous mycobacteria (NTM) in cystic fibrosis (CF) patients. However, it is still a matter of discussion whether criteria for diagnosis of NTM pulmonary infection established by the American Thoracic Society (ATS) are applicable for CF patients. We determined incidence and prevalence of NTM in CF patients and non-CF patients without HIV infection, and validity of ATS criteria for CF patients. Over a period of 2 years, 1,251 respiratory samples were investigated for mycobacteria from 91 CF and 162 non-CF patients. For all patients with NTM recovery, we reviewed clinical charts and determined outcome for up to 2 years. Incidence and prevalence for repeated recovery of NTM were higher in CF patients, but not significantly. CF patients with repeated recovery of NTM met clinical and bacteriological ATS criteria, but radiographic criteria were not met. Treated CF patients showed favorable clinical outcomes, as opposed to untreated patients. We propose a modified definition for diagnosis and hence treatment of NTM pulmonary infection in CF patients.     

Polymorphisms of TGF-beta1 in cystic fibrosis patients

There is a significant phenotypic variance among cystic fibrosis (CF) patients. Due to the role of TGF-beta1 in fibrotic processes we investigated its role in CF pathogenesis. TGF-beta 1 codons 10 and 25 were genotyped in 118 Czech CF patients and 268 controls by PCR-ARMS. Difference between CF and controls was found at codon 10, lower frequency of T/T homozygotes, and codon 25, higher frequency of G/C heterozygotes. We did not prove the association of TGF-beta1 polymorphisms and lung function in CF, however, the TT (codon 10)/GG (codon 25) genotype was preferentially associated with CF-related liver disease and diabetes. Independent of the TGF-beta1 genotype, production of cytokine was higher in patients than in controls with the notable exception of very low levels in Burkholderia cepacia complex colonized patients. In CF, both extremes, highest or lowest TGF-beta 1 production, were associated with impaired lung function.