A range of anaerobic species are present in large numbers in the lungs of patients with CF. If these anaerobic bacteria are contributing significantly to infection and inflammation in the CF lung, informed alterations to antibiotic treatment to target anaerobes, in addition to the primary infecting pathogens, may improve management.

The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured.

A colorimetric assay based on the reduction of a tetrazolium salt {2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT)} for rapidly determining the susceptibility of Pseudomonas aeruginosa isolates to bactericidal antibiotics is described. There was excellent agreement between the tobramycin and ofloxacin MICs determined after 5 h using the XTT assay and after 18 h using conventional methods. The data suggests that an XTT-based assay could provide a useful method for rapidly determining the susceptibility of P. aeruginosa to bactericidal antibiotics.The significant increase in the number of antibiotic-resistant microorganisms causing clinical infection highlights the need for rapid and accurate antimicrobial susceptibility tests. Current standard methods, such as the broth microdilution methods approved by the National Committee for Clinical Laboratory Science (12) and the British Society for Antimicrobial Chemotherapy (BSAC) (1), require at least 18 h before the final antimicrobial susceptibility results are known. When a patient presents with a severe life-threatening infection such as Pseudomonas aeruginosa bacteremia in clinical practice, antibiotic treatment is usually commenced prior to obtaining these results to give the patient the best chance of recovery. Such treatment usually involves the administration of broad-spectrum antibiotics which may be inappropriate (7). The development of rapid susceptibility tests would allow specific and cheaper narrow-spectrum antibiotics to be used, resulting in improved patient care and cost effectiveness and a decrease in the development of antibiotic resistance.Promising alternative colorimetric methods have been proposed as rapid methods for antifungal susceptibility testing of yeasts (8,(13)(14)(15) and Aspergillus species (9-11) and for detection of drug resistance in Mycobacterium tuberculosis (4-6). These methods utilize the reduction of a tetrazolium salt {2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT)} by metabolically active cells to a coloredwater-soluble formazan derivative that can be easily quantified colorimetrically. In the study described here, we developed an XTT-based assay for antimicrobial susceptibility testing which provided results within 5 h. The assay was used to determine the susceptibility of clinical P. aeruginosa isolates to a range of antibiotics, and the drug MICs obtained after 5 h using this method were compared with the MICs obtained after 18 h using the conventional NCCLS and BSAC broth microdilution methods.An initial set of experiments were performed with 4A, a clinical P. aeruginosa isolate, to optimize the quantities of XTT (Sigma Chemical Co, Dorset, United Kingdom) and menadione (Aldrich, Dorset, United Kingdom) required to quantify bacterial metabolic activity. XTT was prepared as saturated solutions at 0.5 and 1 mg/ml in phosphate-buffered saline, filter sterilized, and stored at Ϫ70°C. Menadione was prepared as a 10 mM stock solution in acetone and stored at Ϫ70°C....

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The 'Streptococcus milleri' group (SMG) has recently been recognized as a contributor to bronchopulmonary disease in cystic fibrosis (CF). Routine detection and quantification is limited by current CF microbiology protocols. McKay agar was developed previously for the semi-selective isolation of this group. Here, McKay agar was validated against a panel of clinical SMG isolates, which revealed improved SMG recovery compared with Columbia blood agar. The effectiveness of this medium was evaluated by appending it to the standard CF sputum microbiology protocols in a clinical laboratory for a 6-month period. All unique colony types were isolated and identified by 16S rRNA gene sequencing. Whilst a wide variety of organisms were isolated, members of the SMG were the most prevalent bacteria cultured, and McKay agar allowed routine quantification of the SMG from 10 3 to .10 8 c.f.u. ml "1 directly from sputum. All members of the SMG were detected [Streptococcus anginosus (40.7 %), Streptococcus intermedius (34.3 %) and Streptococcus constellatus (25 %)] with an overall prevalence rate of 40.6 % in our adult CF population. Without exception, samples where SMG isolates were cultured at 10 7 c.f.u. ml "1 or greater were associated with pulmonary exacerbations. This study demonstrates that McKay agar can be used routinely to quantify the SMG from complex clinical samples.