Early Pseudomonas aeruginosa infection in individuals with cystic fibrosis: is susceptibility testing justified?

OBJECTIVES: To test the presumption that Pseudomonas aeruginosa isolates responsible for initial lung infection in individuals with cystic fibrosis (CF) are invariably susceptible to antipseudomonal agents. METHODS: Antibiotic susceptibility was determined (MIC and Etest) in two populations of P. aeruginosa associated with initial lung infection. Population 1: environmental isolates (n=78). Population 2: clinical isolates responsible for first infection in previously non-infected patients (85 isolates from 85 patients). Susceptibility or resistance was determined using current BSAC guidelines; ninth version (2009). RESULTS: The majority (≥ 90%) of isolates in both bacterial populations were susceptible to the front-line antipseudomonal agents; colistin, ciprofloxacin, tobramycin, ceftazidime, amikacin and meropenem. Up to 10% of isolates were resistant to one or more antibiotics. A single isolate from each population would be defined as resistant to tobramycin based on a breakpoint (>128 mg/L) that has been suggested for use in patients receiving inhaled therapy. CONCLUSIONS: The high prevalence of susceptibility found in P. aeruginosa isolates associated with initial infection contrasts with the high prevalence of resistance found in isolates from chronic CF lung infection. However, susceptibility in early isolates cannot be presumed. Until further data are obtained from clinically based studies, susceptibility tests should continue to be performed to assist the choice of antibiotics for treatment of early infection.

Modular branched neurotensin peptides for tumor target tracing and receptor-mediated therapy: a proof-of-concept.

The aim of this study was to demonstrate that oligo-branched peptides can be effective either for spotlighting tumor cells that overexpress peptide receptors, or for killing them, simply by exchanging the functional moiety coupled to the conserved receptor-targeting core. Tetra-branched peptides containing neurotensin (NT) sequence are described here as selective targeting agents for human colon, pancreas and prostate cancer. Fluorophore-conjugated peptides were used to measure tumor versus healthy tissue binding in human surgical samples, resulting in validation of neurotensin receptors as highly promising tumor-biomarkers. Drug-armed branched peptides were synthesized with different conjugation methods, resulting in uncleavable adducts or drug-releasing molecules. Cytotoxicity on human cell lines from colon (HT-29), pancreas (PANC-1) or prostate (PC-3) carcinoma indicated branched NT conjugated with MTX and 5-FdU as the most active agents on PANC-1 (EC(50) 4.4e-007 M) and HT-29 (1.1e-007 M), respectively. Tetra-branched NT armed with 5-FdU was used for in vivo experiments in HT-29-xenografted mice and produced a 50% reduction in tumor growth with respect to animals treated with the free drug. An unrelated branched peptide carrying the same drug was completely ineffective. In vitro and in vivo results indicated that branched peptides are valuable tools for tumor selective targeting.

Application of multiplex single nucleotide primer extension (mSNuPE) to the identification of bacteria: the Burkholderia cepacia complex case.

Burkholderia cepacia complex (BCC) is characterized by a complex taxonomy constituted by seventeen closely related species of both biotechnological and clinical importance. Several molecular methods have been developed to accurately identify BCC species but simpler and effective strategies for BCC classification are still needed. A single nucleotide primer extension (SNuPE) assay using gyrB as a target gene was developed to identify bacteria belonging to the B. cepacia (BCC) complex. This technique allows the successful detection and distinction of single nucleotide polymorphisms (SNPs) and is effectively applied in routine medical diagnosis since it permits to analyze routinely many samples in a few times. Seven SNuPE primers were designed analyzing the conserved regions of the BCC gyrB sequences currently available in databases. The specificity of the assay was evaluated using reference strains of some BCC species. Data obtained enabled to discriminate bacteria belonging to the species B. multivorans, B. cenocepacia (including bacteria belonging to recA lineages III-A, III-C, and III-D), B. vietnamiensis, B. dolosa, B. ambifaria, B. anthina and B. pyrrocinia. Conversely, identification failed for B. cepacia, B. cenocepacia III-B and B. stabilis. This study demonstrates the efficacy of SNuPE technique for the identification of bacteria characterized by a complex taxonomical organization as BCC bacteria.

Transmission of Burkholderia cepacia complex: evidence for new epidemic clones infecting cystic fibrosis patients in Italy.

To analyze national prevalence, genomovar distribution, and epidemiology of the Burkholderia cepacia complex in Italy, 225 putative B. cepacia complex isolates were obtained from 225 cystic fibrosis (CF) patients attending 18 CF centers. The genomovar status of these isolates was determined by a polyphasic approach, which included whole-cell protein electrophoresis and recA restriction fragment length polymorphism (RFLP) analysis. Two approaches were used to genotype B. cepacia complex isolates: BOX-PCR fingerprinting and pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments. A total of 208 (92%) of 225 isolates belonged to the B. cepacia complex, with Burkholderia cenocepacia as the most prevalent species (61.1%). Clones delineated by PFGE were predominantly linked to a single center; in contrast, BOX-PCR clones were composed of isolates collected either from the same center or from different CF centers and comprised multiple PFGE clusters. Three BOX-PCR clones appeared of special interest. One clone was composed of 17 B. cenocepacia isolates belonging to recA RFLP type H. These isolates were collected from six centers and represented three PFGE clusters. The presence of insertion sequence IS 1363 in all isolates and the comparison with PHDC reference isolates identified this clone as PHDC, an epidemic clone prominent in North American CF patients. The second clone included 22 isolates from eight centers and belonged to recA RFLP type AT. The genomovar status of strains with the latter RFLP type is not known. Most of these isolates belonged to four different PFGE clusters. Finally, a third clone comprised nine B. pyrrocinia isolates belonging to recA RFLP type Se 13. They represented three PFGE clusters and were collected in three CF centers.

Antimicrobial susceptibility and synergistic activity of meropenem against Gram-negative non-fermentative bacteria isolated from cystic fibrosis patients.

The objective of the present study was to evaluate the activity of meropenem (a beta-lactam carbapenem with good bactericidal potency and a very wide spectrum of activity) and of ticarcillin, ceftazidime ciprofloxacin, tobramycin, cefepime, which are the most commonly used antimicrobial agents for treatment of pulmonary infections associated with CF. The effect of these antibiotics was tested on 27 multiresistant strains isolated from 24 CF patients during 2000 and 2001. Furthermore, the in vitro synergistic effect of meropenem in association with the other antibiotics was evaluated. Ciprofloxacin, ticarcillin, meropenem and ceftazidime had the most activity and inhibited 66%, 37%, 36% and 33% of strains respectively. The addition of a second antibiotic to meropenem resulted in a synergistic effect on 5 (18.5%) isolates; on average 2.8 synergistic combinations where determined per strain. Of these 27 isolates, antagonism was observed in 3 (11%) strains (1 antagonistic combination per strain). Our study suggests that selecting effective double antibiotic therapy cannot be made empirically for CF patients infected with Gram-negative multiresistant bacilli. Therefore in vitro methods for testing double antibiotic combinations are mandatory.