Microbiology of cystic fibrosis persons not chronically infected with P. aeruginosa: A quasi-experimental study on two different upper airways' sampling methods.

Background: The upper airways of cystic fibrosis (CF) persons are an evolutionary niche where genetically adapted bacterial strains are selected for lung infection. The microbiological studies conducted up to now on the upper airways are not easily comparable. Methods: Using classical culture methods, we simultaneously studied the microbiological status of upper and lower airways in persons not chronically infected with P. aeruginosa. Each person had a single upper airways sampling and a concomitant lower airways sampling. Lower airways sampling was performed by oropharyngeal swab or sputum collection. Using a quasi-experimental design of study, we evaluated the performance of 2 different upper airways' sampling methods, nasal lavage according to method described by Mainz or nasal lavage with a rhino-set. Pain was measured with appropriate scales. Results: A total of 194 persons were enrolled in this study. Pathogenic flora was found in 128 (6.6%) of 194 upper airways samples and in 164 (84.6%) lower airways samples. A statistically significant difference between the upper airways and the lower airways was found in the isolation of S. aureus and non-fermenter gram negatives. Nasal lavage according to Mainz resulted in the isolation of more non-fermenter gramnegatives than the rhino-set (p < 0.05). No differences were found in the pain caused bythe two methods. Conclusions: In our study population, cultures of the upper airway and lower airway differ in CF persons. In people sampled with nasal lavage according to Mainz more non-fermenter gram negatives were detected than with rhino-set. The two sampling methods were comparable with regard to the caused pain, nasal lavage according to Mainz method being quicker to perform.

Nutritional Status and Circulating Levels of Fat-Soluble Vitamins in Cystic Fibrosis Patients: A Cohort Study and Evaluation of the Effect of CFTR Modulators.

BACKGROUND: Improved therapy in CF has led to an overall improvement in nutritional status. The objectives of our study are: to cross-sectionally assess nutritional status and serum levels of fat-soluble vitamins; to retrospectively evaluate the efficacy of modulators on nutritional status and fat-soluble vitamin levels. METHODS: In patients younger than 2 years of age, we evaluated growth, in patients aged 2-18 years, we assessed BMI z-scores, and in adults, we assessed absolute BMI values. Levels of 25(OH)D, vitamins A, and E were measured. RESULTS: A cross-sectional analysis was conducted on 318 patients, 109 (34.3%) with pancreatic sufficiency. Only three patients were under 2 years old. In 135 patients aged 2-18 years, the median BMI z-score was 0.11, and 5 (3.7%) patients had malnutrition (z-score ≤ 2SD). In 180 adults, the median BMI was 21.8 kg/m2. Overall, 15 (13.7%) males (M) and 18 (25.3%) females (F) were underweight (18 < BMI > 20); 3 (2.7%) M and 5 (7.0%) F had a BMI < 18. Suboptimal 25(OH)D levels were found in patients with pancreatic insufficiency. The prevalence of deficiency of vitamins A and E is low. After one year of treatment with modulators, the increase in BMI was more consistent (M: 1.58 ± 1.25 kg/m2 F: 1.77 ± 1.21 kg/m2) in elexacaftor/tezacaftor/ivacaftor (ETI)-treated patients compared with other modulators, with a significant increase in levels of all fat-soluble vitamins. CONCLUSIONS: Malnutrition is present in a limited number of subjects. The prevalence of subjects with suboptimal 25(OH)D levels is high. ETI showed a beneficial effect on nutritional status and circulating levels of fat-soluble vitamins.

Impact of Pancreatitis-Associated Protein on Newborn Screening Outcomes and Detection of CFTR-Related Metabolic Syndrome (CRMS)/Cystic Fibrosis Screen Positive, Inconclusive Diagnosis (CFSPID): A Monocentric Prospective Pilot Experience.

Pancreatitis-Associated Protein (PAP)-based Cystic Fibrosis (CF) newborn bloodspot screening (NBS) protocols detect less CFTR-Related Metabolic Syndrome (CRMS)/CF Screen Positive, Inconclusive Diagnosis (CFSPID). We prospectively evaluated the impact of PAP as the second step of the CF NBS protocol, before the CFTR genetic analysis, on NBS outcomes and CRMS/CFSPID detection in the Tuscany region, Italy. In parallel to the usual protocol (IRT/DNA, protocol 1), PAP was analyzed in IRT-positive infants (IRT/PAP/DNA, protocol 2) from 1 June 2020 until 31 May 2022. We defined an infant as NBS positive if PAP was >1.8 µg/L for IRT value 99th percentile-100 µg/L or >0.6 µg/L for IRT value >100 µg/L. To increase the positive predictive value (PPV) of protocol 2, we retrospectively lowered the upper IRT range value from 100 to 90 µg/L (modified protocol 2). We identified 8 CF and 13 CRMS/CFSPID with protocol 1, 5 CF and 5 CRMS/CFSPID with protocol 2 and 8 CF and 5 CRMS/CFSPID with modified protocol 2. With the PAP-based protocols, we observed a reduction of sweat tests, healthy carrier detection and a significant increase in PPV to 15.38%. Further data are needed in order to evaluate the outcomes of CRMS/CFSPID after a long follow-up.

Methicillin-resistant Staphylococcus aureus eradication in cystic fibrosis patients: A randomized multicenter study.

BACKGROUND: Few studies, based on a limited number of patients using non-uniform therapeutic protocols, have analyzed Methicillin-resistant Staphylococcus aureus (MRSA) eradication. METHODS: In a randomized multicenter trial conducted on patients with new-onset MRSA infection we evaluated the efficacy of an early eradication treatment (arm A) compared with an observational group (B). Arm A received oral rifampicin and trimethoprim/sulfamethoxazole (21 days). Patients' microbiological status, FEV1, BMI, pulmonary exacerbations and use of antibiotics were assessed. RESULTS: Sixty-one patients were randomized. Twenty-nine (47.5%) patients were assigned to active arm A and 32 (52.5%) patients to observational arm B. Twenty-nine (47.5%) patients, 10 patients in arm A and 19 in arm B, dropped out of the study. At 6 months MRSA was eradicated in 12 (63.2%) out of 19 patients in arm A while spontaneous clearance was observed in 5 (38.5%) out of 13 patients in arm B. A per-protocol analysis showed a 24.7% difference in the proportion of MRSA clearance between the two groups (z = 1.37, P(Z>z) = 0.08). Twenty-seven patients, 15 (78.9%) out of 19 in arm A and 12 (92.3%) out of 13 in arm B, were able to perform spirometry. The mean (±SD) FEV1 change from baseline was 7.13% (±14.92) in arm A and -1.16% (±5.25) in arm B (p = 0.08). In the same period the BMI change (mean ±SD) from baseline was 0.54 (±1.33) kg/m2 in arm A and -0.38 (±1.56) kg/m2 in arm B (p = 0.08). At 6 months no statistically significant differences regarding the number of pulmonary exacerbations, days spent in hospital and use of antibiotics were observed between the two arms. CONCLUSIONS: Although the statistical power of the study is limited, we found a 24.7% higher clearance of MRSA in the active arm than in the observational arm at 6 months. Patients in the active arm A also had favorable FEV1 and BMI tendencies.

Chronic infection sustained by a Pseudomonas aeruginosa High-Risk clone producing the VIM-1 metallo-ß-lactamase in a cystic fibrosis patient after lung transplantation.

BACKGROUND: The significance of chronic lung infection by multidrug-resistant (MDR) pathogens in Cystic Fibrosis (CF) transplanted patients remains controversial, and the available information is overall limited. Here we describe the case of a chronic infection, sustained by a metallo-ß-lactamase (MBL)-producing P. aeruginosa strain, in a CF patient following lung transplantation. METHODS: Twelve P. aeruginosa isolates collected from a CF patient over a 15-years follow-up period after lung transplantation were analysed for their antibiotic susceptibility profile, MBL production and clonal relatedness. Available clinical and microbiological records were reviewed. RESULTS: The transplanted CF patient was chronically infected by an MBL-producing P. aeruginosa strain which harboured a blaVIM-1 determinant inserted into a novel class 1 integron. The strain exhibited an MDR phenotype and belonged to the globally widespread ST235 epidemic clonal lineage, which however is not a typical CF-associated epidemic clone. Despite the chronic infection, the long-term outcome of this patient during the post-transplant period was characterized by the absence of acute exacerbations and by a mostly stable pulmonary function. CONCLUSIONS: This report provides one of the few descriptions of MBL-producing P. aeruginosa infections in CF patients, and the first description of such an infection after lung transplantation in these patients. Infection with the MBL-producing strain apparently did not significantly affect the patient pulmonary function.

Essential Oil from Origanum vulgare Completely Inhibits the Growth of Multidrug-Resistant Cystic Fibrosis Pathogens.

Essential oils (EOs) are known to inhibit the growth of a wide range of microorganisms. Particularly interesting is the possible use of EOs to treat multidrug-resistant cystic fibrosis (CF) pathogens. We tested the essential oil (EO) from Origanum vulgare for in vitro antimicrobial activity, against three of the major human opportunistic pathogens responsible for respiratory infections in CF patients; these are methicillin-resistant Staphylococcus aureus, Stenotrophomonas maltophilia and Achromobacter xylosoxidans. Antibiotic susceptibility of each strain was previously tested by the standard disk diffusion method. Most strains were resistant to multiple antibiotics and could be defined as multi-drug-resistant (MDR). The antibacterial activity of O. vulgare EO (OEO) against a panel of 59 bacterial strains was evaluated, with MIC and MBC determined at 24, 48 and 72 hours by a microdilution method. The OEO was effective against all tested strains, although to a different extent. The MBC and MIC of OEO for S. aureus strains were either lower or equal to 0.50%, v/v, for A. xylosoxidans strains were lower or equal to 1% and 0.50%, v/v, respectively; and for S. maltophilia strains were lower or equal to 0.25%, v/v. The results from this study suggest that OEO might exert a role as an antimicrobial in the treatment of CF infections.

Within-host microevolution of Pseudomonas aeruginosa in Italian cystic fibrosis patients.

BACKGROUND: Chronic infection with Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients, and a more complete understanding of P. aeruginosa within-host genomic evolution, transmission, and population genomics may provide a basis for improving intervention strategies. Here, we report the first genomic analysis of P. aeruginosa isolates sampled from Italian CF patients. RESULTS: By genome sequencing of 26 isolates sampled over 19 years from four patients, we elucidated the within-host evolution of clonal lineages in each individual patient. Many of the identified mutations were located in pathoadaptive genes previously associated with host adaptation, and we correlated mutations with changes in CF-relevant phenotypes such as antibiotic resistance. In addition, the genomic analysis revealed that three patients shared the same clone. Furthermore, we compared the genomes of the Italian CF isolates to a panel of genome sequenced strains of P. aeruginosa from other countries. Isolates from two of the Italian lineages belonged to clonal complexes of P. aeruginosa that have previously been identified in Danish CF patients, and our genomic comparison showed that clonal isolates from the same country may be more distantly related than clonal isolates from different countries. CONCLUSIONS: This is the first whole-genome analysis of P. aeruginosa isolated from Italian CF patients, and together with both phenotypic and clinical information this dataset facilitates a more detailed understanding of P. aeruginosa within-host genomic evolution, transmission, and population genomics. We conclude that the evolution of the Italian lineages resembles what has been found in other countries.

Genomes analysis and bacteria identification: The use of overlapping genes as molecular markers.

The growing number of available microbial genomes offers the possibility to identify features that could be used for identification. In this work, the possibility to exploit overlapping genes to develop a simple PCR based method of identification, was explored. Using the Burkholderia cepacia complex as a model, genomic analyses were performed to check the phylogenetic distribution of an overlap between marC and hisH genes and then, a PCR specific for Burkholderia was designed, set up and tested on a panel of strains and on DNA extracted from the sputum of cystic fibrosis patients. Results obtained revealed the usefulness of this approach, which could then be used to develop PCR for the identification of specific bacteria species or genera.

Early antibiotic treatment for Pseudomonas aeruginosa eradication in patients with cystic fibrosis: a randomised multicentre study comparing two different protocols.

BACKGROUND: Pseudomonas aeruginosa chronic pulmonary infection is an unfavourable event in cystic fibrosis. Bacterial clearance is possible with an early antibiotic treatment upon pathogen isolation. Currently, no best practice exists for early treatment. The efficacy of two different regimens against initial P. aeruginosa infection was assessed. METHODS: In a randomised, open-label, parallel-group study involving 13 centres, the superiority of inhaled tobramycin/oral ciprofloxacin compared with inhaled colistin/oral ciprofloxacin (reference treatment) over 28 days was evaluated. Patients were eligible if they were older than 1 year with first or new P. aeruginosa isolation. Treatments were assigned equally by centralised balanced randomisation, stratified by age and forced expiratory volume in 1 s values. The participants and those giving the intervention were not masked to arm assignments. The primary endpoint was P. aeruginosa eradication, defined as three successive negative cultures in 6 months. Analysis was by intention to treat. This trial was registered with EudraCT, number 2008-006502-42. RESULTS: 105 patients were assigned to inhaled colistin/oral ciprofloxacin (arm A) and 118 to inhaled tobramycin/oral ciprofloxacin (arm B). All patients were analysed. P. aeruginosa was eradicated in 66 (62.8%) patients in arm A and in 77 (65.2%) in arm B (OR 0.90, 95% CI 0.52 to 1.55, p=0.81). Following treatment, an increase in Stenotrophomonas maltophilia was noted (OR 3.97, 95% CI 2.27 to 6.94, p=0.001) with no differences between the two arms (OR 0.89, 95% CI 0.44 to 1.78, p=0.88). CONCLUSIONS: No superiority of treatment under study was demonstrated in comparison to the reference treatment. Early eradication treatment was associated with an increase in S maltophilia.

Lactate in cystic fibrosis sputum.

BACKGROUND: Antibiotic therapy is thought to improve lung function in patients with cystic fibrosis (CF) by decreasing neutrophil-derived inflammation. We investigated the origin and clinical significance of lactate in the chronically inflamed CF lung. METHODS: Lactate was measured in sputa of 18 exacerbated and 25 stable CF patients via spectrophotometry and gaschromatography. Lung function was assessed via spirometry. Seven patients with chronic obstructive pulmonary disease (COPD) and three patients with acute lung inflammation served as control groups. Neutrophil and bacterial lactate production was assessed under aerobic and anaerobic conditions. RESULTS: In sputum specimens of patients with respiratory exacerbations lactate concentrations decreased significantly (p<0.005) from 3.4±2.3mmol/L to 1.4±1.4mmol/L after 2-3 weeks of intravenous antibiotics. Successful treatment was reflected in 16 patients (88.9%) by FVC increase associated with lactate decrease (p<0.05). In every single sputum lactate was detectable (3.0±3.1mmol/L, range 0.2-14.1mmol/L). Lactate was lower (1.6±0.8mmol/L) in sputa from seven COPD patients, and it was below the detection limit in three patients with acute lung inflammation. Neutrophil lactate production accumulated up to 10.5mmol/L after 4 days, whereas bacterial lactate production did not appear to contribute substantially to sputum lactate concentrations. CONCLUSIONS: Successful antibiotic therapy is reflected by a decrease in lactate concentrations. Neutrophils are the most likely source for lactate in sputum of CF patients. Therefore lactate may be used to monitor responses to antibiotic therapy as an adjunct to lung function measurements.

Use of the gyrB gene to discriminate among species of the Burkholderia cepacia complex.

Bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can cause serious infections in lungs of cystic fibrosis patients. The Bcc comprises at least nine species that have been discriminated by a polyphasic taxonomic approach. In this study, we focused on the gyrB gene, universally distributed among bacteria, as a new target gene to discriminate among the Bcc species. New PCR primers were designed to amplify a gyrB DNA fragment of about 1900 bp from 76 strains representative of all Bcc species. Nucleotide sequences of PCR products were determined and showed more than 400 polymorphic sites with high sequence similarity values from most isolates of the same species. Phylogenetic tree analysis revealed that most of the 76 gyrB sequences grouped, forming clusters, each corresponding to a given Bcc species.

Patient risk of contact with respiratory pathogens from inanimate surfaces in a cystic fibrosis outpatient clinic. A prospective study over a four-year period.

Acquisition of respiratory pathogens such as Pseudomonas aeruginosa (PA) is associated with increased morbidity and mortality in cystic fibrosis (CF). Research on the prevalence of these pathogens on environmental surfaces of a CF Center is scanty, and so far no study has determined what risk CF patients have of coming in contact with them during their visits to the CF Center. This study is aimed at assessing the prevalence of some respiratory pathogens in samples taken systematically during a 4-year period from inanimate surfaces and sinks in a CF Outpatient Clinic, and to estimate the risk that a non-PA colonized CF patient has of contact with PA when visiting the CF Center. Microbiological samples were taken and cultured from the inanimate surfaces and sinks of the Outpatient clinic of a CF Center once a month from 2001 to 2005. Four hundred and sixty environmental specimens were collected: 36.3% were positive for respiratory pathogens (23% of rooms' inert surfaces, 49.5% of sinks). Achromobacter xylosoxidans was found in 0.8% of surface samples. PA was isolated in 22.8% samples. The estimated risk for each non-colonized patient of coming in contact with PA on the surfaces in the Clinic at each visit was 5.4 per thousand (CI95% 0.9-30.1). Genotyping of a sample of environmental PA strains revealed a genetic relation between environmental and clinical isolates in most cases. Micro-organisms relevant for CF patients can be found on inanimate surfaces of a CF Center, although the risk for patients of coming in contact with PA during their visits to the CF center seems low.

Exopolysaccharides produced by clinical strains belonging to the Burkholderia cepacia complex.

BACKGROUND: In the frame of a research line dedicated to better clarify the role of exopolysaccharides (EPS) in bacterial virulence, EPS produced by species of the Burkholderia cepacia complex (Bcc), namely Burkholderia multivorans, Burkholderia cenocepacia, and a Bcc member of undetermined genomovar, all isolated at the Cystic Fibrosis Regional Centre of Florence (Italy), were investigated for they structural properties. METHODS: Three strains of B. multivorans, three of B. cenocepacia and one of a Bcc member of undetermined genomovar were isolated from CF patients. The reference strains C1576 and J2315, for genomovar II and III, respectively, were included in the study. The bacteria were grown on solid media, the exopolysaccharides produced were purified, and their structures were determined. In addition, sugar analysis of sputum samples was accomplished to search for EPS produced in vivo. RESULTS: Six strains out of seven produced the exopolysaccharide cepacian, while one strain of B. multivorans produced a completely different polymer, previously known in the literature as PS1. Two strains synthesised very small amounts of EPS. No definitive evidence for the presence of cepacian in sputum samples was found. CONCLUSIONS: Most strains examined produced abundant amounts of polysaccharides. Cepacian was the most common EPS isolated and its production was not associated to a particular genomovar.

Breast-feeding in a woman with cystic fibrosis undergoing antibiotic intravenous treatment.

We report the case of a 30-year-old woman with cystic fibrosis (CF) chronically infected with Pseudomonas aeruginosa who delivered and breast-fed a healthy boy. While breast-feeding the woman had to undergo an i.v. antibiotic course with tobramycin, due to pulmonary exacerbation. Tobramycin was not detected in her milk and lactation could be continued. This is the first time that the presence of tobramycin in the milk of a CF woman during i.v. administration has been investigated.