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Inquilinus limosus isolated from a cystic fibrosis patient: first UK report

A variety of unusual Gram-negative, glucose nonfermentative organisms have been isolated from the respiratory secretions of cystic fibrosis (CF) patients, where their pathogenic role is often considered uncertain. Accurate speciation is therefore important as erroneous identification, particularly for Burkholderia cepacia complex (BCC) organisms, can have a significant medical and psychosocial impact for the infected patient, as well as for infection control practices for healthcare workers, hospitals and care centres. Inquilinus limosus is such an organism and is a recently described genus and species (2002), previously only isolated from eight CF patients in the USA, two from Germany and five from France. To date, I. limosus has not been reported in CF patients from either the UK or Ireland. A Gram-negative mucoid, non-fermenting rod was cultured from the sputum of a nineyear-old girl with CF. This colonial morphotype had been cultured on 12 occasions over a two-year period, during which time the patient had remained clinically well. The child had been treated with three monthly maintenance courses of intravenous antipseudomonal antibiotics (usually ceftazidime and gentamicin) and with bi-monthly courses of nebulised tobramycin. The isolate had been cultured from routine sputum specimens obtained at presentation to her CF clinic on B. cepacia selective agar (BCSA; Mast Diagnostics, Bootle, UK) containing ticarcillin, polymyxin and phenol red. The organism produced a characteristic and very mucoid red colony on BCSA, not typical of B. cenocepacia. It was identified as a catalase-positive, oxidase-positive aerobic Gram-negative rod, which grew at 37 ̊C and 42 ̊C. All isolates failed to ferment glucose. Routine biochemical identification was attempted on the isolates using the API 20NE system (bioMérieux, France). However, unacceptable biochemical profiles were obtained, which included the highest score for Agrobacterium radiobacter (API 20NE biochemical profile 1467744 and 1227744, with percentage identifications of 99.8% and 90.2%, respectively). Further phenotypic analysis employing fatty acid analysis showed a very low similarity of the isolate to Ochrobactrum anthropi. Using antibiotic disc sensitivity tests, isolates were consistently colistin-resistant, but were initially sensitive to ceftazidime, ticarcillin, ciprofloxacin, carbenicillin and aminoglycosides when tested on Iso-Sensitest agar. However, antibiotic-resistant variants were noted when a single colony type was retested on blood Iso-Sensitest agar. Subsequently, definite tobramycin resistance emerged on repeat disc testing. Given the relative difficulties experienced with the phenotypic identification of the organism, the isolate was forwarded for molecular identification by polymerase chain reaction (PCR) amplification and direct sequencing of a large but partial region of the 16S ribosomal RNA (rRNA) gene, corresponding to base positions of approximately 257–1304 of Stenotrophomonas maltophilia 16S rRNA (GenBank accession number AY169434). All DNA isolation procedures were carried out in accordance with the DNA contamination management guidelines of Millar et al. and in a class II biological safety cabinet (MicroFlow, England) in a room physically separated from that used to set up nucleic acid amplification reaction mixes and also from the ‘post-PCR’ room, in order to minimise contamination and hence the possibility of falsepositive results. Bacterial DNA was extracted from the isolate using the Roche High Purity PCR Template Preparation Kit (Roche, England) in accordance with the manufacturer ’s instructions. Extracted DNA was transferred to a clean tube and stored at –80 ̊C prior to PCR. For each batch of extractions, a negative extraction control containing all reagents but no isolate was performed. All reaction mixes were set up in a PCR hood. Reaction mixes (50 μL) were set up as follows: 10 mmol/L Tris–HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl2, 200 μM (each) dATP, dCTP, dGTP and dTTP, 1.25 units of Thermus aquaticus (Taq) DNA polymerase (Amplitaq, Perkin Elmer), 0.2 μmol/L of the appropriate broad-range primers XB1 (forward: 5’–CAG ACT CCT ACG GGA GGC AGC AGT -3’) and XB4 (reverse: 5’–GTG TGT ACA AGG CCC GGG AAC 3’) and 4 μL DNA template. Prior to PCR cycling, sealed tubes containing DNA template and all PCR reagents were introduced to the thermal cycler at 96 ̊C to avoid nonspecific annealing during the initial ramp stage. The reaction mixtures were subjected to the following thermal cycling parameters in a Perkin Elmer Cetus 2400 thermocycler: 96 ̊C for 3 min followed by 40 cycles of 96 ̊C for 1 min, 55 ̊C for 1min, 72 ̊C for 1 min, followed by a final extension at 72 ̊C for 10 min. During each run, molecular grade water was included randomly as negative controls and DNA template from Staphylococcus aureus was included as a positive control, as appropriate. For each batch of extractions, an extraction control containing all reagents minus bacterial organism, was performed. Following amplification, 15-μL samples were removed from each reaction mixture and examined by electrophoresis (80 V, 45 min) in gels composed of 2% (w/v) agarose (Gibco, UK) in TAE buffer (40 mmol/L Tris, 20 mmol/L acetic acid, 1 mmol/L EDTA, [pH 8.3]), stained with ethidium bromide (5 μg/100 mL). Gels were visualised under ultraviolet (UV) illumination using a gel image analysis system (UVP Products, England), and all images were archived as digital graphic (*.bmp) files. Amplicons chosen for automated sequencing were purified using a QIAquick PCR purification kit (Qiagen, UK) and eluted in Tris–HCl (10 mmol/L; pH 8.5) prior to sequencing to remove dNTPS, polymerases, salts and Correspondence to: Dr. R. P. D. Cooke