Characterization of Staphylococcus aureus small colony variant strains isolated from Italian patients attending a regional cystic fibrosis care centre.

Small colony variant (SCV) Staphylococcus aureus are a subpopulation of auxotroph, slow-growing strains causing persisting and relapsing infections in cystic fibrosis (CF) patients. Twenty-eight SCV and 29 normal S. aureus strains were isolated from 42 out of 222 Italian CF patients. The isolates were characterized for: susceptibility to antibiotics, methicillin-resistance (MR), Panton Valentine leukocidin, auxotrophy, hypermutability and biofilm formation. Clonal identity of SCV and normal strains was determined by pulsed-field gel electrophoresis. We found that 27 out of 28 SCVs were thymidine-dependent. Furthermore, in contrast to normal phenotype, SCVs were characterized by antibiotic resistance. We also found that 39.3% SCV vs 20.7% normal strains were strong mutators. Moreover, SCVs showed a higher capability to form biofilm compared to normal strains (100% vs 59%). Importantly, we found evidence of clonal spread of SCV strain among CF patients. Using molecular typing, we found that five patients shared the same type A and five out of seven MR-SCV belonged to the same clone (Clone C). The particular virulence and spreading ability of MR-SCV observed highlights the importance of accurate identification and susceptibility testing of these strains. It is important to adopt the optimal approach to treat patients and to prevent cross-infection in CF centres.

First report of chronic pulmonary infection by KPC-3-producing and colistin-resistant Klebsiella pneumoniae sequence type 258 (ST258) in an adult patient with cystic fibrosis.

The spread of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae continues to increase, and the possible development of KPC-producing K. pneumoniae infections in cystic fibrosis (CF) patients is a matter of concern. Here, we describe the establishment of a chronic lung infection due to a colistin-resistant KPC-producing K. pneumoniae isolate in an Italian CF patient.

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.

Antimicrobial use and Pseudomonas aeruginosa susceptibility profile in a cystic fibrosis centre.

The susceptibility patterns of 1315 mucoid and non-mucoid Pseudomonas aeruginosa strains from 224 patients were determined along with antibiotic utilisation in a Cystic Fibrosis Centre from 1993 to 1997. Ceftazidime was the most active agent (86.0% sensitive isolates), followed by piperacillin-tazobactam (81.7%), aztreonam (80.3%), imipenem (80%), piperacillin (76.8%), tobramycin (76.5%), ciprofloxacin (73.7%), ticarcillin (72.4%), ticarcillin-clavulanic acid (70.2%), amikacin (69.5%), netilmicin (56.5%), meropenem (79%) and imipenem (75.5%). The most frequently used compounds were nebulized colistin (mean+/-S.D., 109+/-45 defined daily doses per 1000 patients per day), followed by ciprofloxacin (98+/-8), tobramycin (55+/-9), ceftazidime (31+/-8) and amikacin (55+/-9). The mean antibiotic consumption by our CF patients was 413+/-47 defined daily doses per 1000 patients per day. Trend testing showed a significant decline of susceptibility to aminoglycosides, imipenem and ciprofloxacin, while the susceptibility of P. aeruginosa to piperacillin and ceftazidime was stable.

Exopolysaccharides produced by Burkholderia cenocepacia recA lineages IIIA and IIIB.

Clinical and environmental strains of Burkholderia cenocepacia belonging to the recA lineages IIIA and IIIB were examined for exopolysaccharide (EPS) production. The exopolysaccharides structure was determined using mainly gas chromatography coupled to mass spectrometry and NMR spectroscopy. All the strains produced Cepacian, a highly branched polysaccharide constituted of a heptasaccharide repeating unit, composed of one rhamnose, one glucose, one glucuronic acid, one mannose and three galactose residues. This polymer is the most common exopolysaccharide produced by strains of the Burkholderia cepacia (Bcc) complex. One clinical strain produced also another polysaccharide constituted of three galactose units and one 3-deoxy-D-manno-2-octulosonic acid residues, a polymer that was previously isolated from two strains of B. cepacia genomovar I and B. cenocepacia IIIA.

Epidemiology and clinical course of Burkholderia cepacia complex infections, particularly those caused by different Burkholderia cenocepacia strains, among patients attending an Italian Cystic Fibrosis Center.

In this study, the epidemiology of Burkholderia cepacia complex (Bcc) recovered from the sputum of 75 patients attending the Genoa Cystic Fibrosis (CF) Center at the Gaslini Children's Hospital (Genoa, Italy) was investigated, and the clinical course of the CF patients infected with the different species and genomovars of Bcc was evaluated. All isolates were analyzed for genomovar status by recA gene polymorphism and subsequently random amplified polymorphic DNA fingerprinting. Burkholderia cenocepacia is the predominant species recovered from the CF patients infected with Bcc at the Genoa CF Center. Of the other eight species comprising the Bcc, only a few isolates belonging to B. cepacia genomovar I, Burkholderia stabilis, and Burkholderia pyrrocinia were found. Of the four recA lineages of B. cenocepacia, most patients were infected by epidemic strains belonging to lineages IIIA and IIID, whereas only a few patients harbored IIIB strains. Patient-to-patient spread of Bcc among CF patients was mostly associated with B. cenocepacia, in particular with strains belonging to recA lineages IIIA and IIID. The mortality of CF patients infected with Bcc at the Genoa CF Center was significantly higher than mortality among CF patients not infected with Bcc. All of the deaths were associated with the presence of B. cenocepacia, except the case of a patient infected with B. cepacia genomovar I. Within B. cenocepacia, infection with epidemic strains belonging to lineages IIIA and IIID was associated with higher rates of mortality than was infection with lineage IIIB strains. No significant differences in lung function, body weight, and mortality rate were observed between patients infected with epidemic strains belonging to either B. cenocepacia IIIA or B. cenocepacia IIID.

Burkholderia cepacia complex bacteria from clinical and environmental sources in Italy: genomovar status and distribution of traits related to virulence and transmissibility.

Sixty-eight Burkholderia cepacia complex isolates recovered from the sputum of 53 cystic fibrosis patients and 75 isolates collected from the maize rhizosphere were compared to each other to assess their genomovar status as well as some traits related to virulence such as antibiotic susceptibility, proteolytic and hemolytic activities, and transmissibility, in which transmissibility is determined by detection of the esmR and cblA genes. Among the clinical isolates, B. cepacia genomovar III comprised the majority of isolates examined and only a very few isolates were assigned to B. cepacia genomovar I, B. stabilis, and B. pyrrocinia; among the environmental isolates a prevalence of B. cepacia genomovar III and B. ambifaria was observed, whereas few environmental isolates belonging to B. cepacia genomovar I and B. pyrrocinia were found. Antibiotic resistance analysis revealed a certain degree of differentiation between clinical and environmental isolates. Proteolytic activity and onion tissue maceration ability were found to be spread equally among both clinical and environmental isolates, whereas larger percentages of environmental isolates than clinical isolates had hemolytic activity. The esmR gene was found exclusively among isolates belonging to B. cepacia genomovar III, with a marked prevalence in clinical isolates, whereas only one clinical isolate belonging to B. cepacia genomovar III was found to bear the cblA gene. In conclusion, the results of the present study show that the species compositions of the clinical and environmental B. cepacia complex populations examined are quite different and that some of the candidate determinants related to virulence and transmissibility are not confined solely to clinical isolates but are also spread among environmental isolates belonging to different species of the B. cepacia complex.