Acquired resistance to macrolides in Pseudomonas aeruginosa from cystic fibrosis patients.

Cystic fibrosis (CF) patients receive chronic treatment with macrolides for their antivirulence and anti-inflammatory properties. We, however, previously showed that Pseudomonas aeruginosa, considered as naturally resistant to macrolides, becomes susceptible when tested in a eukaryotic medium rather than a conventional broth.We therefore looked for specific macrolide resistance determinants in 333 CF isolates from four European CF centres in comparison with 48 isolates from patients suffering from hospital-acquired pneumonia (HAP).Minimum inhibitory concentrations (MICs) of macrolides and ketolides measured in eukaryotic medium (RPMI-1640) were higher towards CF than HAP isolates. Gene sequencing revealed mutations at three positions (2045, 2046 and 2598) in domain V of 23S rRNA of 43% of sequenced CF isolates, but none in HAP isolates. Enzymes degrading extracellular polymeric substances also reduced MICs, highlighting a role of the mucoid, biofilm-forming phenotype in resistance. An association between high MICs and chronic azithromycin administration was evidenced, which was statistically significant for patients infected by the Liverpool Epidemic Strain.Thus, ribosomal mutations are highly prevalent in CF isolates and may spread in epidemic clones, arguing for prudent use of oral macrolides in these patients. Measuring MICs in RPMI-1640 could be easily implemented in microbiology laboratories to phenotypically detect resistance.

Antimicrobial Susceptibility of Pseudomonas aeruginosa Isolated from Cystic Fibrosis Patients in Northern Europe.

Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. This study compared the antimicrobial susceptibilities of 153 P. aeruginosa isolates from the United Kingdom (UK) (n = 58), Belgium (n = 44), and Germany (n = 51) collected from 118 patients during routine visits over the period from 2006 to 2012. MICs were measured by broth microdilution. Genes encoding extended-spectrum ß-lactamases (ESBL), metallo-ß-lactamases, and carbapenemases were detected by PCR. Pulsed-field gel electrophoresis and multilocus sequence typing were performed on isolates resistant to ≥3 antibiotic classes among the penicillins/cephalosporins, carbapenems, fluoroquinolones, aminoglycosides, and polymyxins. Based on EUCAST/CLSI breakpoints, susceptibility rates were ≤30%/≤40% (penicillins, ceftazidime, amikacin, and ciprofloxacin), 44 to 48%/48 to 63% (carbapenems), 72%/72% (tobramycin), and 92%/78% (colistin) independent of patient age. Sixty percent of strains were multidrug resistant (MDR; European Centre for Disease Prevention and Control criteria). Genes encoding the most prevalent ESBL (BEL, PER, GES, VEB, CTX-M, TEM, SHV, and OXA), metallo-ß-lactamases (VIM, IMP, and NDM), or carbapenemases (OXA-48 and KPC) were not detected. The Liverpool epidemic strain (LES) was prevalent in UK isolates only (75% of MDR isolates). Four MDR sequence type 958 (ST958) isolates were found to be spread over the three countries. The other MDR clones were evidenced in ≤3 isolates and localized in a single country. A new sequence type (ST2254) was discovered in one MDR isolate in Germany. Clonal and nonclonal isolates with different susceptibility profiles were found in 20 patients. Thus, resistance and MDR are highly prevalent in routine isolates from 3 countries, with meropenem, tobramycin, and colistin remaining the most active drugs.

Carrier-free combination for dry powder inhalation of antibiotics in the treatment of lung infections in cystic fibrosis.

The aim of the study was to develop an efficient combination antibiotic formulation containing tobramycin and clarithromycin as a dry powder for inhalation. A carrier-free formulation of the two drugs was produced by spray-drying and characterised for its aerodynamic behaviour by impaction tests with an NGI and release profiles. The particle size distribution, morphological evaluation and crystallinity state were determined by laser diffraction, scanning electron microscopy and powder X-ray diffraction, respectively. Drug deposition profiles were similar for the two antibiotics, which has a synergistic effect, allowing them to reach the target simultaneously at the expected dose. The release profiles show that tobramycin and clarithromycin should probably dissolve without any difficulties in vivo in the lung as 95% of tobramycin and 57% of clarithromycin mass dissolved in 10min for the spray-dried formulation. The FPF increased from 35% and 31% for the physical blend for tobramycin and clarithromycin, respectively, to 65% and 63% for the spray-dried formulation. The spray-dried formulation shows particularly high deposition results, even at sub-optimal inspiratory flow rates, and therefore, represents an attractive alternative in the local treatment of lung infection such as in cystic fibrosis.

Efficacy of the combination of tobramycin and a macrolide in an in vitro Pseudomonas aeruginosa mature biofilm model.

Respiratory disease is the main cause of morbidity and mortality in patients with cystic fibrosis (CF). In particular, patients suffer from chronic infection due to biofilm formation by opportunistic Pseudomonas aeruginosa (32). Therefore, there is an urgent need to develop alternative ways to treat biofilm-associated clinical infections. The aim of this study was to compare the antimicrobial effects in vitro of the combinations tobramycin-clarithromycin and tobramycin-azithromycin against five P. aeruginosa biofilms and to establish the most effective combination. We performed a kinetic study over a period of 28 days of a twice-daily coadministration of the combinations tobramycin-clarithromycin and tobramycin-azithromycin on 12-day-old, mature P. aeruginosa biofilms formed on microplate pegs for 4 clinical isolates and one laboratory strain (PAO1) to simulate the treatment of CF patients with tobramycin inhalation solution (TOBI) through aerosolization. A synergy between tobramycin and clarithromycin was recorded for 3/5 biofilms, with a bacterial decrease of more than 5 log. Conversely, we found an antagonistic activity when 4 µg/ml tobramycin was administered with azithromycin at 2 µg/ml for P. aeruginosa PAO1 and with azithromycin at 2, 20, 50, 100, and 200 µg/ml for P. aeruginosa PYO1. Treatment with tobramycin at 4 µg/ml combined with clarithromycin at 200 µg/ml eradicated all five biofilms, while tobramycin-azithromycin at the same concentrations eradicated only three biofilms. Results of this study suggest that local administration of tobramycin and clarithromycin into the respiratory tract represents a better strategy than the combination tobramycin-azithromycin for the treatment of P. aeruginosa-associated pulmonary infections.

Efficacy of artesunate + sulfamethoxypyrazine/pyrimethamine versus praziquantel in the treatment of Schistosoma haematobium in children.

BACKGROUND: This study was conducted to determine the efficacy of the antimalarial artemisinin-based combination therapy (ACT) artesunate +sulfamethoxypyrazine/pyrimethamine (As+SMP), administered in doses used for malaria, to treat Schistosoma haematobium in school aged children. METHODOLOGY/PRINCIPAL FINDINGS: The study was conducted in Djalakorodji, a peri-urban area of Bamako, Mali, using a double blind setup in which As+SMP was compared with praziquantel (PZQ). Urine samples were examined for Schistosoma haematobium on days -1, 0, 28 and 29. Detection of haematuria, and haematological and biochemical exams were conducted on day 0 and day 28. Clinical exams were performed on days 0, 1, 2, and 28. A total of 800 children were included in the trial. The cure rate obtained without viability testing was 43.9% in the As+SMP group versus 53% in the PZQ group (Chi(2) = 6.44, p = 0.011). Egg reduction rates were 95.6% with PZQ in comparison with 92.8% with As+SMP, p = 0.096. The proportion of participants who experienced adverse events related to the medication was 0.5% (2/400) in As+SMP treated children compared to 2.3% (9/399) in the PZQ group (p = 0.033). Abdominal pain and vomiting were the most frequent adverse events in both treatment arms. All adverse events were categorized as mild. CONCLUSIONS/SIGNIFICANCE: The study demonstrates that PZQ was more effective than As+SMP for treating Schistosoma haematobium. However, the safety and tolerability profile of As+SMP was similar to that seen with PZQ. Our findings suggest that further investigations seem justifiable to determine the dose/efficacy/safety pattern of As+SMP in the treatment of Schistosoma infections. TRIAL REGISTRATION: ClinicalTrials.gov NCT00510159.

Evaluation of long-term co-administration of tobramycin and clarithromycin in a mature biofilm model of cystic fibrosis clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa colonisation and chronic lung infection associated with biofilm formation is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. There is thus an urgent need to develop alternative ways to treat biofilm-associated clinical infections. A kinetic study of twice-daily co-administration of the antibiotics tobramycin and clarithromycin was performed over 28 days on 12-day-old mature P. aeruginosa biofilms formed on microplate pegs for 23 clinical isolates of various phenotypes and genotypes to simulate the treatment of CF patients with inhaled tobramycin through aerosolisation (TOBI). Drug activity was assessed by enumeration of persistent bacteria before, during and after treatment. A mature (12-day-old) biofilm model was chosen because a previous study suggested that such a biofilm was a more realistic in vitro model than a 24-h-old biofilm. Synergistic activity of the drug combination was confirmed on biofilms of 9/23 P. aeruginosa isolates. Of these nine isolates, total destruction of the biofilm was observed for five of them. Combination treatment was superior or equivalent to treatment with tobramycin alone, as activity was observed on 47.8% of the isolates with the combination versus 26.1% with tobramycin alone. No correlation was observed between drug susceptibility profiles and the phenotype or genotype of the isolates.

Effect of antibiotic co-administration on young and mature biofilms of cystic fibrosis clinical isolates: the importance of the biofilm model.

The prognosis of patients with cystic fibrosis (CF) has improved dramatically over the last three decades although the majority of patients still die in early adulthood. Infection with Pseudomonas aeruginosa has generally been associated with declining lung function and increased mortality in patients. This study aimed to investigate the in vitro activity of tobramycin/clarithromycin combination on biofilms of clinical isolates of P. aeruginosa, meticillin-susceptible and -resistant Staphylococcus aureus, and Burkholderia cepacia. First, the impact of antibiotic co-administration on biofilms at different stages of maturation, i.e. during early formation and on 24-h-old and 12-day-old biofilms, was compared. The 24-h-old biofilms were found to behave differently compared with those aged 12 days, which were more resistant to antibiotics. A kinetic study of antibiotic co-administration twice a day for 9 days on 12-day-old P. aeruginosa biofilms was then performed to simulate the effect of treatment of CF patients by inhaled tobramycin through aerosolisation (TOBI). The results obtained support a synergistic activity of tobramycin/clarithromycin combination on biofilms of P. aeruginosa PY02 and PA01, with a logarithmic bacterial decrease of 3.37 and 3.96, respectively. On the other hand, increased resistance to each of the antibacterial agents used alone was observed. This study highlights the importance of the biofilm stage for in vitro investigations and enabled the development of an in vitro model of mature biofilm that is more appropriate to mimic in vivo conditions in CF patients.

In vitro activity of antibiotic combinations against Pseudomonas aeruginosa biofilm and planktonic cultures.

In order to investigate the hypothesis that the combination of clarithromycin with other antibacterial agents offers a successful treatment in the eradication of Pseudomonas aeruginosa biofilm, we first determined the activity of eight antimicrobial agents against planktonic cultures of P. aeruginosa isolates by the microdilution technique. Second, we determined the in vitro effects of these antimicrobial agents individually and in combination against planktonic cultures and pre-formed biofilms of P. aeruginosa PA01. Drug combinations with marked activity were tested on biofilms of clinical isolates. The percentages of planktonic culture survival and biofilm persistence were determined using spectrophotometry and scanning electron microscopy, respectively. Bacterial enumeration was used as a more quantitative method to assess the viability of bacteria in the biofilm. Among the antibacterial agents tested, tobramycin and polymyxin B had the strongest activity against planktonic cultures when tested alone. Synergistic activity was observed for the combination chitosan/tobramycin against planktonic cultures but not biofilms, and for the combination tobramycin/clarithromycin against biofilms but not planktonic cultures. The results suggest that the combination clarithromycin/tobramycin may be successful for eradicating infections involving bacterial biofilms such as in cystic fibrosis patients chronically infected by P. aeruginosa. Further studies on representative isolates in vivo are warranted to support these results.