Genomovar diversity amongst Burkholderia cepacia complex isolates from an Australian adult cystic fibrosis unit

In this study, a combination of recA-based PCR assays and 16S rDNA restriction fragment length polymorphism (RFLP) analysis was used to determine the genomovar diversity of clinical Burkholderia cepacia complex isolates. Twenty-eight isolates were prospectively collected from patients attending a large Australian adult cystic fibrosis (CF) unit, 22 isolates were referred from other Australian CF units and a further eight isolates originated from patients without CF. The 28 prospectively collected isolates were distributed amongst the following genomovars: Burkholderia cepacia genomovar I (28.6%), Burkholderia multivorans (21.4%), Burkholderia cepacia genomovar III (39.3%), Burkholderia vietnamiensis (3.6%) and Burkholderia ambifaria (7.1%). The results of this study highlight the usefulness of 16S rDNA RFLP typing for the identification of other Burkholderia spp. and non-fermenting gram-negative bacteria.

Antibiotic desensitization in adults with cystic fibrosis

Objective: Allergic reactions to antibiotics occur in up to 30% of patients with cystic fibrosis (CF). Repeated antibiotic exposure and immune hyper-responsiveness increase the risk of allergic reactions and may limit antibiotic choice. Desensitization may allow the successful administration of an antibiotic despite previous allergy. We aimed to determine the success of antibiotic desensitization in patients with CF in an adult CF unit over a 7-year period. Methodology: A retrospective medical record review was performed on the 19 patients who had undergone antibiotic desensitization procedures. Data collected included drug allergy and intolerance profiles, nature of allergies, and the outcome of desensitization procedures. Desensitization procedures were performed in a ward setting according to published methods. Results: Nineteen patients (13 females) reported 62 drug allergies with a mean of 3.3 per patient. Of the 71 desensitization procedures undergone by this group, 54 (76%) were successful. Fifteen of the 19 patients were allergic to two or more beta-lactam antibiotics. Over half of the patients were desensitized to more than one antibiotic. Nine different antibiotics were used in 31 different patient/drug combinations. A successful outcome was achieved in 18/31 (58%) combinations, with three requiring treatment for mild allergic reactions. Allergic reactions caused drug cessation in a total of 19 patient/drug combinations (three after initial successful desensitization and full courses of antibiotics). Over 50% of these reactions occurred on day 1. Desensitization failures were more common in patients with well-documented allergic reactions to a specific drug. Conclusion: This study demonstrates that multiple antibiotic allergies are common in adults with CE Cross-reactivity between beta-lactam antibiotics may limit antibiotic choice for the treatment of pulmonary exacerbations. Antibiotic desensitization allows safe and successful treatment in the ward setting of many patients with previous allergies to an antibiotic. In many patients symptoms of allergy still occur and result in cessation of the antibiotics. Use of corticosteroids and antihistamines may improve the success rate of desensitization procedures.

The use of 16s rDNA restriction fragment length polymorphism analysis for the identification of non-fermenting gram negative bacilli in a cystic fibrosis microbiology referral service

The utility of 16s rDNA restriction fragment length polymorphism (RFLP) analysis for the partial genomovar differentiation of Burkholderia cepacia complex bacterium is well documented. We compared the 16s rDNA RFLP signatures for a number of non-fermenting gram negative bacilli (NF GNB) LMG control strains and clinical isolates pertaining to the genera Burkholderia, Pseudomonas, Achromobacter (Alcaligenes), Ralstonia, Stenotrophomonas and Pandoraea. A collection of 24 control strain (LMG) and 25 clinical isolates were included in the study. Using conventional PCR, a 1.2 kbp 16s rDNA fragment was generated for each organism. Following restriction digestion and electrophoresis, each clinical isolate RFLP signature was compared to those of the control strain panel. Nineteen different RFLP signatures were detected from the 28 control strains included in the study. TwentyoneyTwenty- five of the clinical isolates could be classified by RFLP analysis into a single genus and species when compared to the patterns produced by the control strain panel. Four clinical B. pseudomallei isolates produced RFLP signatures which were indistinguishable from B. cepacia genomovars I, III and VIII. The identity of these four isolates were confirmed using B. pseudomallei specific PCR. 16s rDNA RFLP analysis can be a useful identification strategy when applied to NF GNB, particularly for those which exhibit colistin sulfate resistance. The use of this molecular based methodology has proved very useful in the setting of a CF referral laboratory particularly when utilised in conjunction with B. cepacia complex and genomovar specific PCR techniques. Species specific PCR or sequence analysis should be considered for selected isolates; especially where discrepancies between epidemiology, phenotypic and genotypic characteristics occur.

Molecular epidemiology of Aspergillus collected from cystic fibrosis patients

Background - Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. Methods - Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing. Results - Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. Conclusions - To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.

Use of selective medium for Burkholderia cepacia isolation in respiratory samples from cystic fibrosis patients

Burkholderia cepacia colonizes cystic fibrosis (CF) patients. We evaluated the impact of the use of a selective medium in the rate of B. cepacia recovery from respiratory samples of CF patients. During a 6-month period, respiratory samples were collected from 106 CF patients and cultivated on selective media including a B. cepacia selective medium. Confirmation of the identity of B. cepacia isolates was carried out by species specific PCR and determination of genomovar status performed by a sequential PCR approach. Results of B. cepacia isolation during this period were compared to the preceding two years, when the sample processing was identical except for the lack of the B. cepacia selective medium. B. cepacia was isolated in 11/257 (4.2%) of the samples using the selective medium, in contrast with the preceding two years, when it was isolated in 6/1029 samples (0.58%), p < 0.0001. Identity of all 11 isolates was confirmed by PCR and genomovar determination was accomplished in all but one isolate. These results suggest that the use of a selective medium increases recovery rate of B. cepacia from respiratory samples.

Comparison of five methods for oxacillin susceptibility testing of Staphylococcus aureus isolates from cystic fibrosis patients

Methicillin-resistant Staphylococcus aureus (MRSA) are now a worldwide problem. Cystic fibrosis (CF) patients are commonly colonized and infected by MRSA. Accurate oxacillin susceptibility testing is mandatory for the adequate management of these patients. We performed a comparison of the accuracy of different tests in CF isolates, including methicillin-susceptible S. aureus and MRSA with different SCCmec types, and using the mecA gene as the gold-standard. The sensitivity and specificity of oxacillin disc, Etest, and oxacillin agar screening plate were 100%. Sensitivity of the cefoxitin disc was 85% and specificity was 100%. For clinically relevant isolates, laboratories may consider the use of a combination of two phenotypic methods.

Cystic Fibrosis, Atopy, Asthma and ABPA

The role of atopy on cystic fibrosis (CF) progression remains unclear but evidence suggests that it may influence the appearance of co-morbid conditions such as CF asthma or allergic bronchopulmonary aspergillosis (ABPA). Recognising asthma in patients with CF is not always easy but the identification of atopic markers favours the diagnosis. Physicians should be aware of this fact in order to achieve a better control of respiratory symptoms in patients with CF. Bronchial mucosa inflammation and abnormal mucus predispose to mould colonisation. These patients are at higher risk of allergic sensitisation, especially when atopic susceptibility is present. In the particular case of A. fumigatus, allergic sensitisation precedes ABPA development, which occurs in up to 10% of CF patients. Progression of lung function deterioration is most strikingly pronounced in patients with ABPA. Therefore, sensitisation with A. fumigatus should be regularly tested in patients with CF, especially those at higher risk. Recombinant allergens constitute an important advance in differentiating Aspergillus sensitisation from ABPA itself.

High prevalence of methicillin-resistant Staphylococcus aureus with SCCmec type III in cystic fibrosis patients in southern, Brazil

INTRODUCTION: Bacterial colonization of the lungs is the main cause of morbidity in cystic fibrosis (CF). Pathogens such as Staphylococcus aureus are very well adapted to the pulmonary environment and may persist for years in the same patient. Genetic determinants of these bacteria, such as the presence of SCCmec have recently emerged as a problem in this population of patients. METHODS: Staphylococcus aureus isolates obtained from different clinical materials coming from CF and non-CF patients attended at a cystic fibrosis reference hospital were compared according to SCCmec type and antibiotic susceptibility profile. RESULTS: Three hundred and sixty-four single-patient Staphylococcus aureus isolates were collected, of which 164 (45%) were from CF patients. Among the latter, 57/164 (44.5%) were MRSA, and among the non-CF patients, 89/200 (35%) were MRSA. Associated pathogens were found in 38 CF patients. All 57 MRSA from CF patients harbored the multiresistant cassette type III. In contrast, 31/89 MRSA from non-CF patients harbored SCCmec type I (35%) and 44/89 harbored type III (49%). The antibiotic susceptibility pattern was similar between CF and non-CF patients. CONCLUSIONS: The high prevalence of multiresistant SCCmec type III among CF patients compared with non-CF patients in our institution may make it difficult to control disease progression through antibiotic therapy for promoting the survival of this kind of patient.