A single multilocus sequence typing (MLST) scheme was developed for precise characterization of the opportunistic pathogens of Burkholderia cepacia complex (BCC), a group composed of at least nine closely related species. Seven conserved housekeeping genes were selected after a comparison of five Burkholderia species, and a collection of strains was subjected to nucleotide sequence analysis using a nested PCR amplification approach for each gene. MLST differentiated all nine current BCC species and identified 114 sequence types within a collection of 119 strains. No differentiation was found between strains recovered from environmental or clinical sources. The improved resolution in strain identification offered by MLST was able to identify previously characterized epidemic strain lineages and also demonstrated the presence of four novel potential species groups within the complex. There was also evidence for recombination having an important role in the recent evolution of individual BCC species. This highly transferable, validated, MLST scheme provides a new means to assist in species identification as well as unambiguous strain discrimination of the BCC by a single approach. It is also the first MLST scheme designed at the outset to incorporate multiple species and should facilitate global epidemiological investigations of the BCC.

Background and Purpose-Several studies have implied an association between Chlamydia pneumoniae and atherosclerosis. Our research was designed to investigate the association of this organism with strokes and transient cerebral ischemia. Methods-Antibodies to C pneumoniae were measured in 176 patients with stroke or transient cerebral ischemia and 1518 control subjects with noncardiovascular, nonpulmonary disorders. Acute infection or reinfection was defined by IgG Ն512 or IgM Ն8 or fourfold rise in IgG, and previous infection was defined by IgG 64 to 256 or lgA Ն8. Logistic regression was used to examine the influences of ethnic origin, age, sex, smoking habit, diabetes mellitus, steroid medication, and social deprivation on antibody levels. Some patients underwent CT and carotid ultrasound examinations and cholesterol, triglyceride, fibrinogen, and von Willebrand factor estimations. Results-We found that 13.6% of stroke/transient ischemic attack (TIA) patients and 5.7% of control subjects had antibody titers suggesting acute C pneumoniae (re)infection, while 32.4% of stroke/TIA patients and 12.7% of control subjects had titers suggesting previous infection (PϽ.05). Stroke/TIA patients differed from control subjects in their levels of acute and previous infection, with adjusted odds ratios of 4.2 (95% CI, 2.5 to 7.1) and 4.4 (95% CI, 3.0 to 6.5), respectively. These did not differ notably between strokes resulting from major nonhemorrhagic infarcts, small-vessel infarcts, or hemorrhage. Cholesterol, triglyceride, fibrinogen, and von Willebrand factor concentrations showed no apparent association with titers. Conclusions-These data support the association of cerebral vascular disease with previous C pneumoniae infection and the association of stroke and transient cerebral ischemia with recrudescence of infection. (Stroke. 1998;29:404-410.)

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Chronic obstructive pulmonary disease (COPD) is characterised by sputum production, bacterial colonisation, neutrophilic bronchial airway inflammation and poor health status. The aim of this study was to determine the impact of sputum potentially pathogenic microorganisms (PPMs) on bronchial airway inflammation, health status and plasma fibrinogen levels in subjects with moderate-to-severe COPD during the clinical stable state.Sputum total cell and neutrophil counts, supernatant interleukin-8, leukotriene B 4 , tumour necrosis factor-a and neutrophil elastase levels, neutrophil chemotaxis and plasma fibrinogen levels were estimated. Health status was determined using the St George9s Respiratory Questionnaire and the 36-item Short-Form Health Survey questionnaire.Twenty-seven (40%) subjects had PPMs and 40 (60%) non-PPMs in their sputum. Both groups were of similar age, body mass index, smoking history and lung function. The PPMs group showed significantly higher levels of interleukin-8, leukotriene B 4 , tumour necrosis factor-a, neutrophil elastase and increased neutrophil chemotaxis. They also exhibited worse health status and raised plasma fibrinogen levels compared to the non-PPMs group.In conclusion, subjects with clinically stable moderate-to-severe chronic obstructive pulmonary disease who had potentially pathogenic microorganisms in their sputum demonstrated an exaggerated airway inflammatory response, poorer health status and increased plasma fibrinogen levels than those who had nonpotentially pathogenic microorganisms. Eur Respir J 2004; 23: 685-691.

Pseudomonas aeruginosa produces quorum sensing signal molecules that are potential biomarkers for infection.A prospective study of 60 cystic fibrosis patients with chronic P. aeruginosa, who required intravenous antibiotics for pulmonary exacerbations, was undertaken. Clinical measurements and biological samples were obtained at the start and end of the treatment period. Additional data were available for 29 of these patients when they were clinically stable.Cross-sectionally, quorum sensing signal molecules were detectable in the sputum, plasma and urine of 86%, 75% and 83% patients, respectively. They were positively correlated between the three biofluids. Positive correlations were observed for most quorum sensing signal molecules in sputum, plasma and urine, with quantitative measures of pulmonary P. aeruginosa load at the start of a pulmonary exacerbation. Plasma concentrations of 2-nonyl-4-hydroxy-quinoline (NHQ) were significantly higher at the start of a pulmonary exacerbation compared to clinical stability (p<0.01). Following the administration of systemic antibiotics, plasma 2-heptyl-4-hydroxyquinoline (p=0.02) and NHQ concentrations (p<0.01) decreased significantly.In conclusion, quorum sensing signal molecules are detectable in cystic fibrosis patients with pulmonary P. aeruginosa infection and are positively correlated with quantitative measures of P. aeruginosa. NHQ correlates with clinical status and has potential as a novel biomarker for P. aeruginosa infection.

Members of the Burkholderia cepacia complex (Bcc), found in many environments, are associated with clinical infections. Examining diverse species and strains from different environments with multilocus sequence typing, we identified >20% of 381 clinical isolates as indistinguishable from those in the environment. This finding links the natural environment with the emergence of many Bcc infections.

The concentrations of moxifloxacin achieved after a single 400 mg dose were measured in serum, epithelial lining fluid (ELF), alveolar macrophages (AM) and bronchial mucosa (BM). Concentrations were determined using a microbiological assay. Nineteen patients undergoing fibre-optic bronchoscopy were studied. Mean serum, ELF, AM and BM concentrations at 2.2, 12 and 24 h were as follows: 2.2 h: 3.2 mg/L, 20.7 mg/L, 56.7 mg/L, 5.4 mg/kg; 12 h: 1.1 mg/L, 5.9 mg/L, 54.1 mg/L, 2.0 mg/kg; 24 h: 0.5 mg/L, 3.6 mg/L, 35.9 mg/L, 1.1 mg/kg, respectively. These concentrations exceed the MIC(90)s for common respiratory pathogens such as Streptococcus pneumoniae (0.25 mg/L), Haemophilus influenzae (0.03 mg/L), Moraxella catarrhalis (0.12 mg/L), Chlamydia pneumoniae (0.12 mg/L) and Mycoplasma pneumoniae (0. 12 mg/L) and indicate that moxifloxacin should be effective in the treatment of community-acquired, lower respiratory tract infections.

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