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Background: The advent of CFTR modulators highlighted that the sweat test (ST) for CF can be used also as an outcome measure for the basic defect of CFTR. Despite the technological advances, ST still remains operator-dependent and its execution should be strongly paired with guidelines. In 2022, due to the advent of CFTR modulators, the Italian CF Society introduced a specific ST report. The aim of the present paper is to discuss the impact of this new report in the 2022-23 round of the Italian External Quality Assessment program for ST (I-EQA-SCT). Methods: The scheme of the I-EQA-SCT is prospective, enrolment is voluntary, the payment of a fee is required and results are shared through a web-facility. Assessment covers analysis, interpretation, and reporting of results. In the 2022-23 round, 2 out of the 3 mock clinical information referred to patients who started modulators. Results: Fourteen laboratories completed the 2022-23 I-EQA-SCT round. Three of them failed in the interpretation of results from these two mock cases and/or used a wrong report not consistent with the more recent Italian Sweat Test Recommendations. Conclusions: The overall results obtained from the laboratories involved in the I-EQA-SCT program clearly showed that the laboratories’ qualitative and quantitative performance improved significantly. Results emerged from this round highlighted an issue in the report form used for monitoring patients on CFTR modulator therapy thus stressing the importance of these programs in improving both the performance of lab services and ameliorating the sweat test recommendations.

Key Clinical Message The introduction of newborn screening for cystic fibrosis (CF) increased diagnosis of cystic fibrosis screen positive inconclusive diagnosis (CFSPID). We described the case of a 12‐month‐old boy with CFSPID who, during summer, presented Pseudo‐Bartter syndrome with no diagnostic criteria for CF.

Cystic fibrosis (CF) is a rare autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation is F508del-CFTR (ΔF) which leads the encoded ion channel towards misfolding and premature degradation. The disease is characterized by chronic bronchopulmonary obstruction, inflammation and airways colonization by bacteria, which are the major cause of morbidity and mortality. The STING pathway is the main signaling route activated in the presence of both self and pathogen DNA, leading to Type I Interferon (IFN I) production and the innate immune response. In this study, we show for the first time the relationship existing in CF between resistant and recurrent opportunistic infections by Pseudomonas aeruginosa and the innate immunity impairment. We demonstrate through ex vivo and in vivo experiments that the pathway is inadequately activated in ΔF condition and the use of direct STING agonists, as 2′,3′-cyclic GMP-AMP (2’, 3’ cGAMP), is able to restore the immune response against bacterial colonization. Indeed, upon treatment with the STING pathway agonists, we found a reduction of colony forming units (CFUs) consequent to IFN-β enhanced production in Pseudomonas aeruginosa infected bone marrow derived macrophages and lung tissues from mice affected by Cystic Fibrosis. Importantly, we also verified that the impairment detected in the primary PBMCs obtained from ΔF patients can be corrected by 2’, 3’ cGAMP. Our work indicates that the cGAS/STING pathway integrity is crucial in the Cystic Fibrosis response against pathogens and that the restoration of the pathway by 2’, 3’ cGAMP could be exploited as a possible new target for the symptomatic treatment of the disease.

Abstract Background Cystic Fibrosis Related Diabetes (CFRD) is a frequent comorbidity of patients with Cystic Fibrosis (CF). A worsening of clinical conditions appears before CFRD. It has been demonstrated a decline in pulmonary function and nutritional status also in patients with prediabetes. Few trials show that insulin may be beneficial in prediabetic CF patients, to date guidelines do not recommend for this condition. Case presentation We report a case of a patient treated with insulin glargine at 13 years, due to glycemic intolerance, and with Lumacaftor/Ivacaftor at 15 years. A reduction of pulmonary exacerbations was observed after glargine therapy, also confirmed after the starting of Lumacaftor/ Ivacaftor in this patient. Pulmonary function improved only after the first year of glargine therapy, then a deterioration appeared due to the natural history of CF lung damage. During the COVID-19 lockdown, poor adherence to care contributed to diabetes mellitus onset needing high insulin requirements. After two weeks the patient returned to prediabetic condition and his previous dose of glargine. Conclusions our case highlights firstly that insulin glargine has contributed to preserve him from further clinical worsening due to prediabetes in the years before pandemic, secondly the negative impact of COVID-19 lockdown on the clinical course of a chronic disease as CF.

Abstract Background The goal of mucoactive therapies in cystic fibrosis (CF) is to enhance sputum clearance and to reduce a progressive decline in lung function over the patient's lifetime. We aimed to investigate the level of consensus among specialists from Italian CF Centers on appropriateness of therapeutic use of dornase alfa (rhDNase) for CF patients. Method A consensus on appropriate prescribing in CF mucoactive agents was appraised by an online Delphi method, based on a panel of 27 pulmonologists, coordinated by a Scientific Committee of six experts in medical care of patients with CF. Results Full or very high consensus was reached on several issues related to therapeutic use of dornase alfa for CF patients in clinical practice. Conclusions The consensus reached on a number of topics regarding use of mucoactive agents in patients with CF can help guide clinicians in daily practice based on expert experience and define the most appropriate therapeutic strategy for the individual patient.

Abstract Background The management of chronic diseases, particularly in children, requires an integrated physical and psychological approach to both sick children and their family. This is the case of Cystic Fibrosis (CF), a complex genetic chronic disease, where, a comprehensive evaluation of the emotional impact and an effective multidimensional approach are indicated. Aim This study investigates on parenting stress in children and adolescents with CF and its determinants related to parents, children and the disease severity. Methods The study involved 34.04% adult males and 65.96% adult females (range 21-55 years) and 47 children with CF, 54.35% males and 45.65% females (range 1-17 years). The data were obtained through a Parenting Stress Index – Short Form (PSI-SF) questionnaire. According to the PSI-SF scoring system, three types of stress were detected: a typical stress pattern (normal), a high stress pattern (increased) and a defensive response, which may be considered as a high stress feature in children which requires monitoring and clinical evaluation. Results This study shows a significant presence of stress in females (60.23%), of subject married (84.62%), unemployed (69.23%) and with education level such as “middle School” (61.54%). Concerning children of parents with high stress, it resulted most frequent children with one sibling (53.85%). Finally, by univariate analysis, it resulted a significant positive correlation between parenting stress and disease degree of children. Instead by multivariate analysis, we found that the variables: Number of siblings and Birth order were a significant positive and negative predictor of parenting stress respectively. Conclusion An increased stress level was detected in less than one third of parents of subjects with CF. These data may be related to the psychological support which is part of the routine management of CF care team. However, as children’s features seem to act as a determinant of stress more than parental ones, the parental-child dysfunction should be the target for further integrated interventions.

Abstract Virtual Reality (VR) as a tool for pain reduction is the research topic of several clinical trial for Randomized Controlled Trials despite its wide use in the daily clinical practice for non- pharmacological reduction of pain in some countries. At present, there are no published reviews of VR-efficacy of pain reduction in pediatric patients. That is why we made a systematic review of the efficacy of VR as a tool for pain reduction in children and adolescents. Electronic databases and gray literature published between 2014 and 2019 were analyzed. A total of 9 studies were eligible according to the established inclusion criteria. Results show that virtual reality is a valid tool for non-pharmacological pain reduction and that this approach is to be preferred to the standard reduction techniques currently in use. However, more studies using standardized experimental methodologies are needed to provide more systematic comparison and quantitative synthesis.

Abstract Familial loss-of-function mutations of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) channel protein cause cystic fibrosis (CF), the most frequent inherited life-threatening disease in the Caucasian population. A recent study indicates that the gluten/gliadin-derived peptide (P31–43) can cause CFTR inhibition in intestinal epithelial cells, thus causing a local stress response that contributes to the immunopathology of celiac disease (CD). Accordingly, an increased prevalence of CD has been observed in several cohorts of CF patients. CD is characterized by a permanent intolerance to gluten/gliadin proteins occurring in a proportion of susceptible individuals who bear the human leukocyte antigen (HLA) DQ2/DQ8. In CD, perturbations of the intestinal environment, together with the activation of the innate immune system by P31–43, are essential for rendering other immunodominant gliadin peptide fully antigenic, thus triggering an adaptive immune response with an autoimmune component. P31–43-induced CFTR inhibition elicits the danger signals that ignite the epithelial stress response and perturb epithelial proteostasis. Importantly, potentiators of CFTR channel gating, such as the FDA-approved drug Ivacaftor, prevent P31–43 driven CFTR inhibition and suppress the gliadin-induced stress response in cells from celiac patients, as well as the immunopathology developing in gliadin-sensitive mice. Thus, CFTR potentiators may represent a novel therapeutic option for celiac patients.

Abstract Background A clinical heterogeneity was reported in patients with Cystic Fibrosis (CF) with the same CFTR genotype and between siblings with CF. Methods We investigated all clinical aspects in a cohort of 101 pairs of siblings with CF (including 6 triplets) followed since diagnosis. Results Severe lung disease had a 22.2% concordance in sib-pairs, occurred early and the FEV1% at 12 years was predictive of the severity of lung disease in the adulthood. Similarly, CF liver disease occurred early (median: 15 years) and showed a concordance of 27.8% in sib-pairs suggesting a scarce contribution of genetic factors; in fact, only 2/15 patients with liver disease in discordant sib-pairs had a deficiency of alpha-1-antitrypsin (a known modifier gene of CF liver phenotype). CF related diabetes was found in 22 pairs (in 6 in both the siblings). It occurred later (median: 32.5 years) and is strongly associated with liver disease. Colonization by P. aeruginosa and nasal polyposis that required surgery had a concordance > 50% in sib-pairs and were poorly correlated to other clinical parameters. The pancreatic status was highly concordant in pairs of siblings (i.e., 95.1%) but a different pancreatic status was observed in patients with the same CFTR mutations. This suggests a close relationship of the pancreatic status with the “whole” CFTR genotype, including mutations in regulatory regions that may modulate the levels of CFTR expression. Finally, a severe course of CF was evident in a number of patients with pancreatic sufficiency. Conclusions Physicians involved in care of patients with CF and in genetic counseling must be aware of the clinical heterogeneity of CF even in sib-pairs that, at the state of the art, is difficult to explain.

This study aims to investigate cholesterol metabolism in a mouse model with cystic fibrosis (CF) by the comparison of affected homozygous versus wild type (WT) mice. In particular, we evaluated the effects of a diet enriched with cholesterol in both mice groups in comparison with the normal diet. To this purpose, beyond serum and liver cholesterol, we analyzed serum phytosterols as indirect markers of intestinal absorption of cholesterol, liver lathosterol as indirect marker of de novo cholesterol synthesis, liver cholestanol (a catabolite of bile salts synthesis) and the liver mRNA levels of LDL receptor (LDLR), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR), acyl CoA:cholesterol acyl transferase 2 (ACAT2), cytochrome P450 7A1 (CYP7A1) and tumor necrosis factor alpha (TNFα). CF mice showed lower intestinal absorption and higher liver synthesis of cholesterol than WT mice. In WT mice, the cholesterol supplementation inhibits the synthesis of liver cholesterol and enhances its catabolism, while in CF mice we did not observe a reduction of LDLR and HMG-CoAR expression (probably due to an altered feed-back), causing an increase of intracellular cholesterol. In addition, we observed a further increase (5-fold) in TNFα mRNA levels. This preliminary study suggests that in CF mice there is a vicious circle in which the altered synthesis/secretion of bile salts may reduce the digestion/absorption of cholesterol. As a result, the liver increases the biosynthesis of cholesterol that accumulates in the cells, triggering inflammation and further compromising the metabolism of bile salts.

Abstract Background Recurrent (RP) and chronic pancreatitis (CP) may complicate Cystic Fibrosis (CF). It is still unknown if mutations in genes involved in the intrapancreatic activation of trypsin (IPAT) or in the pancreatic secretion pathway (PSP) may enhance the risk for RP/CP in patients with CF. Methods We enrolled: 48 patients affected by CF complicated by RP/CP and, as controls 35 patients with CF without pancreatitis and 80 unrelated healthy subjects. We tested a panel of 8 genes involved in the IPAT, i.e. PRSS1, PRSS2, SPINK1, CTRC, CASR, CFTR, CTSB and KRT8 and 23 additional genes implicated in the PSP. Results We found 14/48 patients (29.2%) with mutations in genes involved in IPAT in the group of CF patients with RP/CP, while mutations in such genes were found in 2/35 (5.7%) patients with CF without pancreatitis and in 3/80 (3.8%) healthy subjects (p

Summary: Candida albicans is implicated in intestinal diseases. Identifying host signatures that discriminate between the pathogenic versus commensal nature of this human commensal is clinically relevant. In the present study, we identify IL-9 and mast cells (MCs) as key players of Candida commensalism and pathogenicity. By inducing TGF-β in stromal MCs, IL-9 pivotally contributes to mucosal immune tolerance via the indoleamine 2,3-dioxygenase enzyme. However, Candida-driven IL-9 and mucosal MCs also contribute to barrier function loss, dissemination, and inflammation in experimental leaky gut models and are upregulated in patients with celiac disease. Inflammatory dysbiosis occurs with IL-9 and MC deficiency, indicating that the activity of IL-9 and MCs may go beyond host immunity to include regulation of the microbiota. Thus, the output of the IL-9/MC axis is highly contextual during Candida colonization and reveals how host immunity and the microbiota finely tune Candida behavior in the gut. : Deciphering the mechanisms by which Candida albicans promotes either pathology or protective tolerance in the gut could be clinically relevant. Renga et al. show a key role for IL-9 and mast cells in promoting either inflammatory dysbiosis and pathology or tolerance in leaky gut models and human celiac disease. Keywords: IL-9, mast cells, Candida albicans, intestinal inflammation, IDO1, celiac disease

Abstract Background An increasing number of patients have been described as having a number of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) variants for which it lacks a clear genotype–phenotype correlation. We assesses the clinical features of patients bearing the S737F (p.Ser737Phe) CFTR missense variant and evaluated the residual function of CFTR protein on nasal epithelial cells (NEC). Methods A retrospective database was performed from individuals homozygous or compound heterozygous for the S737F variant followed in the Cystic Fibrosis (CF) Centre of Florence. We performed a nasal brushing in cooperating patients and compared the results with those of patients followed in the pediatric CF Centre of Naples. Results 9/295 (3%) subjects carrying at least S737F CFTR variant on one allele were identified. Patients were diagnosed in 7/9 cases by newborn screening and in two cases for dehydration with hypochloremic metabolic alkalosis; at diagnosis sweat chloride levels (SCL) were in the pathological range in only one case. After a mean follow up of 8,6 years (range 0,5–15,8), SCL were in the pathological range in 8/9 cases (mean age at CF diagnosis: 1,5 years), all patients were pancreatic sufficiency and respiratory function was normal. The gating activity on NEC was 15.6% and 12.7% in two patients compound heterozygous for W1282X and DelE22_24, while it was ranged between 6,2% and 9,8% in CF patients. Conclusions S737F is a CFTR mutation associated to hypochloremic alkalosis in childhood, mild CF phenotype in teenage years and a residual function of CFTR protein.

Abstract Background Primary ciliary dyskinesia (PCD) and cystic fibrosis (CF) are increasingly compared. There are no chest magnetic resonance imaging (MRI) comparative studies of PCD and CF. We assessed clinical, functional, microbiological and MRI findings in PCD and mild CF patients in order to evaluate different expression of lung disease. Methods Twenty PCD (15.1 years) and 20 CF subjects with mild respiratory impairment (16 years, 70% with pancreatic insufficiency) underwent MRI, spirometry, and sputum cultures when clinically stable. MRI was scored using the modified Helbich system. Results PCD was diagnosed later than CF (9.9 versus 0.6 years, p = 0.03), despite earlier symptoms (0.1 versus 0.6 years, p = 0.02). In the year preceding the study, patients from both groups underwent two systemic antibiotic courses (p = 0.48). MRI total scores were 11.6 ± 0.7 and 9.1 ± 1 in PCD and CF, respectively. FEV1 and FVC Z-scores were −1.75 (range, −4.6–0.7) and −0.6 (−3.9–1.8) in PCD, and −0.9 (range, −5.4–2.3) and −0.3 (−3.4–2.5) in CF, respectively. No difference was found between lung function or structure, despite a higher MRI subscore of collapse/consolidation in PCD versus CF (1.6 ± 0.1 and 0.6 ± 0.2, p

BackgroundFew studies, based on a limited number of patients using non-uniform therapeutic protocols, have analyzed Methicillin-resistant Staphylococcus aureus (MRSA) eradication.MethodsIn 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.ResultsSixty-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.ConclusionsAlthough 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.

Cystic fibrosis (CF) is a lifelong disorder affecting 1 in 3500 live births worldwide. It is a monogenetic autosomal recessive disease caused by loss-of-function mutations in the gene encoding the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR), the impairment of which leads to ionic disequilibria in exocrine organs. This translates into a chronic multisystemic disease characterized by airway obstruction, respiratory infections, and pancreatic insufficiency as well as hepatobiliary and gastrointestinal dysfunction. Molecular characterization of the mutational heterogeneity of CFTR (affected by more than 2000 variants) improved the understanding and management of CF. However, these CFTR variants are linked to different clinical manifestations and phenotypes, and they affect response to treatments. Expanding evidence suggests that multisystemic disease affects CF pathology via impairing either CFTR or proteins regulated by CFTR. Thus, altering the expression of miRNAs in vivo could constitute an appealing strategy for developing new CF therapies. In this review, we will first describe the pathophysiology and clinical management of CF. Then, we will summarize the current knowledge on altered miRNAs in CF patients, with a focus on the miRNAs involved in the deregulation of CFTR and in the modulation of inflammation. We will highlight recent findings on the potential utility of measuring circulating miRNAs in CF as diagnostic, prognostic, and predictive biomarkers. Finally, we will provide an overview on potential miRNA-based therapeutic approaches.

Background: Newborn screening (NBS) early-identifies cystic fibrosis (CF), but in CF-screening positive inconclusive diagnosis (CF-SPID) the results of immunoreactive trypsinogen (IRT), molecular analysis and sweat test (ST) are discordant. A percentage of CF-SPID evolves to CF, but data on long-term monitoring are lacking. We describe the follow-up of all CF and CF-SPID identified between 2008 and 2019. Methods: NBS was performed by IRT followed by molecular analysis and ST between 2008 and 2014; double IRT followed by molecular analysis and ST after 2014. Results: NBS revealed 47 CF and 99 CF-SPID newborn, a ratio 1:2.1—the highest reported so far. This depends on the identification by gene sequencing of the second variant with undefined effect in 40 CF-SPID that otherwise would have been defined as carriers. Clinical complications and pulmonary infections occurred more frequently among CF patients than among CF-SPID. Two CF-SPID cases evolved to CF (at two years), while eight evolved to CFTR-related disorders (CFTR-RD), between one and eight years, with bronchiectasis (two), recurrent pneumonia (four, two with sinonasal complications), recurrent pancreatitis (two). No clinical, biochemical or imaging data predicted the evolution. Conclusion: Gene sequencing within the NBS reveals a higher number of CF-SPID and we first describe an approach to early identify CFTR-RD, with relevant impact on their outcome.

About 50% of patients with cystic fibrosis (CF) have sinonasal complications, which include inferior turbinate hypertrophy (NTH) and/or nasal polyposis (NP), and different degrees of lung disease, which represents the main cause of mortality. Monitoring of sinonasal disease requires complex instrumental procedures, while monitoring of lung inflammation requires invasive collection of bronchoalveolar lavage fluid. The aim of this study was to investigate the associations between salivary cytokines levels and CF-related airway diseases. Salivary biochemical parameters and cytokines, i.e., interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNF-α), were analyzed in resting saliva from healthy subjects and patients with CF. Patients with CF showed significantly higher levels of salivary chloride, IL-6, IL-8, and TNF-α and lower calcium levels than healthy subjects. Among patients with CF, IL-6 and IL-8 were significantly higher in patients with NTH, while TNF-α was significantly lower in patients with NP. A decreasing trend of TNF-α in patients with severe lung disease was also observed. On the other hand, we did not find significant correlation between cytokine levels and Pseudomonas aeruginosa or Stenotrophomonas maltophilia colonization. These preliminary results suggest that salivary IL-6 and IL-8 levels increase during the acute phase of sinonasal disease (i.e., NTH), while the end stages of pulmonary disease and sinonasal disease (i.e., NP) show decreased TNF-α levels.

Intestinal inflammation is a hallmark of cystic fibrosis (CF). Administration of probiotics can reduce intestinal inflammation and the incidence of pulmonary exacerbations. We investigated the composition of intestinal microbiota in children with CF and analyzed its relationship with intestinal inflammation. We also investigated the microflora structure before and after Lactobacillus GG (LGG) administration in children with CF with and without antibiotic treatment.The intestinal microbiota were analyzed by denaturing gradient gel electrophoresis (DGGE), real-time polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH). Intestinal inflammation was assessed by measuring fecal calprotectin (CLP) and rectal nitric oxide (rNO) production in children with CF as compared with healthy controls. We then carried out a small double-blind randomized clinical trial with LGG.Twenty-two children with CF children were enrolled in the study (median age, 7 years; range, 2-9 years). Fecal CLP and rNO levels were higher in children with CF than in healthy controls (184±146 µg/g vs. 52±46 µg/g; 18±15 vs. 2.6±1.2 µmol/L NO2 (-), respectively; P

Bacilli belonged to genus Kluyvera are Gram negative non-fermentative microorganisms described in the Enterobacteriaceae family, generally considered as saprophytes of the digestive tract.Their role in pathogenesis of infectious disease is unclear, but recently, they are emerged as a cause of clinically significant disease in no-CFpopulation and several anatomic sites are involved as urinary, respiratory and gastrointestinal tracts. In the present note, we describe the identification of Kluyvera ascorbata isolates, obtained from sputum samples of one CF patient (male, 10 years old). The note concerns a patient suffering from chronic obstructive bronchopulmonary disease and chronic Pseudomonas aeruginosa infection. Isolates showed a good growth on MacConkey agar and BCSA, after incubation at 37°C for 24-48 h and they were sensitive to several antibiotics, as cephalosporins, carbapenems, quinolones and trimethoprim-sulfamethoxazole. Clinical observation and the spirometric parameters early before and after isolation of microrganism did not show any worsening status in our patient. The selective pressure given by intensive use of antibiotics in CF patients increases susceptibility to infections by opportunistic microrganisms. Our report underlines the importance of careful bacterial identification and antimicrobial susceptibility study in defining the potential role of new pathogen bacteria in CF lung and to address possible therapeutic strategies that may help to guide antibiotic therapy regimes in CF patients.

Pulmonary infections by Gram-negative bacteria such as Achromobacter xylosoxidans are recovered frequently in patients with Cystic Fibrosis. Aims of this study were to value the isolation frequency of A.xylosoxidans strains in a cohort of Cystic Fibrosis patients, to investigate their antimicrobial sensitivity and to establish possible clonal likeness among strains.A retrospective study was undertaken between January 2004 and December 2008 on 300 patients receiving care at the Regional Cystic Fibrosis Centre of “Federico II” University, Naples. Sputum samples were collected and selective media as well as commercial systems for bacterial identification were used. The activity of antimicrobial agents was determined using diffusion and micro-dilution methods. For DNA-fingerprinting, a genomic DNA macrorestriction followed by pulsed-field gel electrophoresis was carried out. A total of 238 strains from 51 patients were isolated. Strains were resistant to aztreonam, about half of these were resistant to gentamicin and trimethoprim-sulphamethoxazole. They were sensitive to piperacillin, piperacillin/tazobactam, and also to carbapenems, quinolones, cephalosporines. Macrorestriction analysis applied on some isolates showed substantial heterogeneity among strains.Actually, the prognostic role of A. xylosoxidans in Cystic Fibrosis is unclear, but this finding must imply difficulties on therapeutic approach. So, it is need to be on the look out regard such microorganisms. Preliminary results of DNA-fingerprinting indicate no evidence of clonal likeness and then of patient-to-patient spread.

Staphylococcus aureus is one of the most common pathogens isolated from respiratory tracts of Cystic Fibrosis patients (CF). The infection by this pathogen starts in early infancy, often preceding chronic infections by Pseudomonas aeruginosa. The infection and colonization by methicillin-resistant Staphylococcus aureus (MRSA) are, by then, events very frequent among CF patients and this bacterial isolation leads to complications in therapeutic management because of the limited treatment options. Strains of Staphylococcus aureus are able to produce biofilms on natural or synthetic surfaces. Biofilms are sophisticated communities of matrix-encased bacteria and infections by biofilm-producing bacteria are particularly problematic because sessile bacteria can often withstand host immune responses and are generally much more tolerant to antibiotics. The first aim of this work is to evaluate the ability of MRSA strains isolated from respiratory secretions of CF patients to develop biofilms in comparison with methicillin-sensitive Staphylococcus aureus (MSSA) strains obtained from respiratory secretions of CF patients.Therefore, our second aim is to evaluate the environmental influence on this ability. To evaluate the development of biofilm on solid matrix and the possible environmental influence,we applied the method described by Christensen et al. We found that a significantly higher number of MRSA strains were biofilm positive compared with MSSA strains (p

Pseudomonas aeruginosa is the leading cause of chronic lung infection and following pulmonary worsening of cystic fibrosis patients. To verify whether bacterial modifications regarding motility, mucoidy, and serum susceptibility proceeded from an adaptation to chronic infection or a replacement with a new strain, sequential P. aeruginosa isolates of known phenotype collected from 5 cystic fibrosis patients were typed by pulsed-field gel electophoresis (PFGE). Antimicrobial susceptibility testing of all isolates was performed by the disc diffusion method. PFGE typing demonstrated that strains dissimilar in colony morphotype and of different antibiotic susceptibility patterns could be of the same genotype. Some patients were colonized with a rather constant P. aeruginosa flora, with strains of different phenotypes but of one genotype. Instead, some patients may be colonized by more than one genotype. Secretion of mucoid exopolysaccharide and acquisition of a new antibiotic susceptibility phenotype in these strain appear to evolve during chronic colonization in cystic fibrosis patients from specific adaptation to infection rather than from acquisition of new bacterial strains.

Pseudomonas aeruginosa is the major pulmonary pathogen that causes morbidity and mortality in burned, immunocompromised and cystic fibrosis patients. Among the various virulence factors, type IV-pili, play a major role in mediating bacteria-host cells interactions, in formation of biofilm and for twiching motility.These pili are composed of pilin, 15000-16000 molecular weight monomeric subunit, synthesized from pilA gene. The N-terminal region of pilin protein is strong conserved and is important for the oligomerization.The C-terminal region is less conserved and contains a disulfide-bonded loop (DSL) structure that is thought to interact with the eukaryotic glycolipid receptor “asialo GM1”. Analysis of pilin allele distribution among isolates from various sources revealed the presence of six groups of pilin allels characterized by different DSL sequence and different associated accessory genes in pilA chromosomal locus. 81 P. aeruginosa isolates were recovered from cystic fibrosis patients during a 3 years period. 30 of these strains were grown and their genomic DNA was prepared using a rapid method for gram-negative bacteria. PCR primers were used for amplification of pilA and adjacent sequences revealing the presence of three different amplification products. One of these is highly homologous with pilA gene of PA14 strain, the others are identical to PA103 and PAK pilA genes. Our study revealed in the prevalence of isolates with group II pilin genes from Cystic Fibrosis compared with other groups that are predominant in previous studies.

Objectives: Pulmonary infections by Gram-negative bacteria, as Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, are the major cause of morbidity in Cystic Fibrosis patients. In the past decade, several pathogens as Alcaligenes spp and no tuberculosis mycobacteria have been recovered in these patients. Bacteria of genus Chryseobacterium are widespread Gram-negative microrganisms and involved in human infections. Aims of this study were to value the isolation frequency of Chryseobacterium strains in a cohort of Cystic Fibrosis patients, to investigate their antimicrobial sensibility and to establish possible clonal likeness between strains. Methods:A retrospective study was undertaken between January 2003 and December 2005 on 300 patients receiving care at the Regional Cystic Fibrosis Centre of Naples University “Federico II”. Sputum samples were checked: for bacterial identification, selective media and commercial identification systems were used.The activity of antimicrobial agents was determined using diffusion and microdiluthion methods. For DNA-fingerprinting, a genomic DNA macrorestriction followed by pulsed-field electrophoresis was carried out. Results:A total of 26 strains from 17 patients were isolated (7 C. meningosepticum, 14 C. indologenes, 5 C. gleum). Strains were resistant to cephalosporins and carbapenems; some were sensitive to ciprofloxacin, levofloxacin and trimethoprim-sulphamethoxazole. Macrorestriction analysis showed substantial heterogeneity among strains. Conclusions: Actually, the prognostic role of Chryseobacterium in Cystic Fibrosis is unclear and although the small number of isolations, it is need to be on the look out regard such microorganisms. The considerable resistance implies difficulties on therapeutic approach. Results of DNA-fingerprinting indicate no evidence of clonal likeness and then of patient-to-patient spread.

Objectives: Cystic Fibrosis patients are prone to infection by Gram-negative bacteria, such as Pseudomonas aeruginosa and Burkholderia cepacia, which become very resistant with recurrent antibiotic treatments.The purpose of this study was to evaluate the susceptibility patterns of 12 isolates of Burkholderia cepacia and 8 isolates of Pseudomonas aeruginosa, isolated from Cystic Fibrosis patients to five individual antibiotics (ceftazidime, ciprofloxacin, piperacillin/tazobactam, levofloxacin and trimethoprim-sulfamethoxazole) and to four antibiotic combinations (ceftazidime associated with one of the other antibiotics). Methods: Susceptibility tests were carried out using an agar diffusion method, the E-test (AB Biodisk, Solna, Sweden). Results: Strains were selected because of their resistance to individual antimicrobial agents, tested with automated system (Phoenix, BD), which ranged from 41.6% for ceftazidime to 83.3% for ciprofloxacin for Burkholderia cepacia and from 25% for ceftazidime to 100% for trimethoprim-sulfamethoxazole for Pseudomonas aeruginosa. By using E-test,we were able to demonstrate synergy against 2 strains of Pseudomonas aeruginosa (25%) with ceftazidime- piperacillin/tazobactam. No synergy was detected against all strains of Burkholderia cepacia. Conclusions:These results suggest that the E-test offers a simple, labour-efficient and accurate method for MIC determination on agar medium and the susceptibility to antibiotic combinations greatly improves the guide to antibiotic therapy for infections to Gram-negative bacteria in Cystic Fibrosis patients.