Investigation of a Pandoraea apista cluster common to adult and paediatric cystic fibrosis patients attending two hospitals in the same city.

PURPOSE: We examined evidence for transmission of Pandorea apista among cystic fibrosis (CF) patients attending paediatric and adult services in one city who had previously been found to harbour related isolates by pulsed-field gel electrophoresis (PFGE). METHODOLOGY: The whole-genome sequences of 18 isolates from this cluster from 15 CF patients were examined, along with 2 cluster isolates from 2 other centres. The annotated sequence of one of these, Pa14367, was examined for virulence factors and antibiotic resistance-associated genes in comparison with data from a 'non-cluster' isolate, Pa16226. RESULTS: Single-nucleotide polymorphism (SNP) analysis suggested that cluster isolates from the same city differed from one another by a minimum of 1 and a maximum of 383 SNPs (an average of 213 SNPs; standard deviation: 18.5), while isolates from the 2 other hospitals differed from these by a minimum of 34 and 61 SNPs, respectively. Pa16226 differed from all cluster isolates by a minimum of 22 706 SNPs. Evidence for patient-to-patient transmission among isolates from the same city was relatively limited, although transmission from a common source could not be excluded. The annotated genomes of Pa14367 and Pa16226 carried putative integrative and conjugative elements (ICEs), coding for type IV secretion systems, and genes associated with heavy metal degradation and carbon dioxide fixation, and a wide selection of genes coding for efflux pumps, beta-lactamases and penicillin-binding proteins. CONCLUSION: Epidemiological analysis suggested that this cluster could not always be attributed to patient-to-patient transmission. The acquisition of ICE-related virulence factors may have had an impact on its prevalence.

rpoB gene sequencing highlights the prevalence of an E. miricola cluster over other Elizabethkingia species among UK cystic fibrosis patients.

Difficulties in distinguishing species of the Elizabethkingia genus by MALDI-TOF prompted use of rpoB sequencing to investigate species distribution among 44 isolates from cystic fibrosis (CF) patients. Forty-three isolates from 38 patients formed a cluster comprising E. miricola and proposed novel species E. bruuniana sp. nov., the exception clustering with proposed species E. ursingii sp. nov., also part of this wider cluster. All 44 isolates were PCR-positive for urease gene ureG, whereas only one of 23 E. anophelis isolates from non-CF patients was positive, suggesting that this gene is largely associated with the E. miricola cluster. Antibiotic susceptibilities of 12 CF isolates revealed all were resistant to beta-lactams with the exception of piperacillin-tazobactam, and were only susceptible to minocycline and co-trimoxazole. Pulsed-field gel electrophoresis analysis revealed 4 shared strains among 17 CF patients in one pediatric clinic, but epidemiological investigations did not support patient-to-patient transmission except between one sibling pair.

Prevalence of Burkholderia species, including members of Burkholderia cepacia complex, among UK cystic and non-cystic fibrosis patients.

PURPOSE: We aimed to establish the prevalence of different Burkholderia species among UK cystic fibrosis (CF) and non-CF patients over a 2 year period. METHODOLOGY: Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to identify isolates to genus level, followed by recA/gyrB sequence clustering or species-specific PCR. In all, 1047 Burkholderia isolates were submitted for identification from 361 CF patients and 112 non-CF patients, 25 from the hospital environment and three from a commercial company. Potential cross-infection was assessed by pulsed-field gel electrophoresis (PFGE) and multi- locus-sequence typing (MLST). MICs were determined for 161 Burkholderia cepacia complex (Bcc) isolates. CF Trust registry data were sought to examine clinical parameters relating to Bcc infection. RESULTS: Burkholderia multivorans was the most prevalent species among CF patients affecting 56 % (192) patients, followed by Burkholderia cenocepacia IIIA (15 %; 52 patients). Five novel recA clusters were found. Among non-CF patients, Burkholderia cepacia was the most prevalent species (37/112; 34 %), with 18 of 40 isolates part of a UK-wide B. cepacia 'cluster'. This and three other clusters were investigated by PFGE and MLST. Cable-pili positive isolates included two novel sequence types and representatives of ET12. Antibiotic susceptibility varied between and within species and CF/non- CF isolates. CF Trust registry data suggested no significant difference in lung function between patients harbouring B. cenocepacia, B. multivorans and other Bcc species (P=0.81). CONCLUSION: The dominance of B. multivorans in CF, the presence of a B. cepacia cluster among non-CF patients and the existence of putative novel species all highlighted the continuing role of Burkholderia species as opportunistic pathogens.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus from bacteraemia in northern Italy.

BACKGROUND: Vancomycin is frequently used in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia; reduced susceptibility to vancomycin is therefore disturbing. METHODS: molecular epidemiological analysis of 81 MRSA bacteraemia isolates collected during 2002-10 in the province of Bolzano, northern Italy was performed. MICs of a range of antimicrobials were determined by agar microdilution, screening for hGISA was by Macro-Etest and Etest GRD and confirmed by PAP-AUC with vancomycin and teicoplanin. All isolates were characterised by toxin gene profiling, agr, spa, and SCCmec-typing; MLST and PFGE were carried out on representative strains. RESULTS: The dominant clones identified were ST8-MRSA-IVc (55%) and ST228- and ST111-MRSA-I (25%); most of the latter two lineages (19/20; 95%) were GISA or PAP-AUC confirmed hGISA. One ST8-MRSA-IVc isolate harboured ccrA2B2 together with ccrA4B4. The remainder were diverse genotypically and belonged to MLST clonal complexes 1, 22, 45 and 398. CONCLUSIONS: Diverse lineages of MRSA were identified as causing bacteraemia in a province in northern Italy. The association of a specific genotype with the hGISA and GISA phenotypes among representatives of the second most common lineage identified is of clinical concern.

Identification of Achromobacter xylosoxidans by detection of the bla(OXA-114-like) gene intrinsic in this species.

Achromobacter xylosoxidans is an emerging pathogen among patients with cystic fibrosis. Here we describe a specific PCR for identification of this organism, based on detection of bla(OXA-114-like). Comparison of isolates by pulsed-field gel electrophoresis revealed evidence of cross-infection in some cases, but most patients harbored their own strain.

Linezolid-resistant ST36 methicillin-resistant Staphylococcus aureus associated with prolonged linezolid treatment in two paediatric cystic fibrosis patients.

OBJECTIVES: To describe the emergence of linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) of sequence type (ST)36 lineage in two paediatric patients with cystic fibrosis, after long-term low-dose linezolid treatment. METHODS: Two paediatric males with cystic fibrosis had sputum samples quantitatively cultured during hospitalization. After the isolation of MRSA from both patients, oral treatment with 300 mg linezolid twice daily was initiated for periods of 1-2 months separated by up to 6 months. Isolates cultured 9 months after the start of treatment were tested for resistance to linezolid by agar dilution (BSAC). Resistant isolates were examined for 23S rDNA mutations, and typed by phage and macrorestriction with SmaI. Isolates from follow-up sputum samples were obtained until 44-51 months after treatment with linezolid. RESULTS: Colonization with MRSA was at a density of approximately 10(6) cfu/mL sputum for both subjects. Initial isolates were susceptible to linezolid, but, 9 months later, isolates from both patients were resistant (MICs > 16 mg/L). Both isolates were epidemic MRSA-16 variant A1 (ST36-MRSA-II), which is widespread in UK hospitals. Both isolates were heterozygous for a G2576T mutation in their 23S rDNA genes, but one was resistant to fusidic acid and tetracycline. In follow-up sampling, the younger patient yielded linezolid-resistant EMRSA-16 for a further 42 months, whilst the other lost the linezolid-resistant MRSA and had alternately Pseudomonas aeruginosa or linezolid-susceptible EMRSA-16 variant A1 isolated over 35 further months. CONCLUSIONS: Linezolid resistance emerged in two isolates of ST36 MRSA colonizing the lungs of two paediatric cystic fibrosis patients. Subtherapeutic levels of linezolid may have facilitated the selection of resistance.