Within 1 h of collection, specimens were spread on blood and MacConkey agar plates and were incubated at 35°C for 18 h. Five colonies of each morphotype were selected for identification. Gram-negative bacteria, includ- ing P. aeruginosa, were identified by using the BBL Crystal ID system (Becton Dickinson, Sparks, MD). Antimicrobial susceptibility testing was performed by the disc-diffusion technique, according to guidelines of the National Committee for Clinical Laboratory Standards (6), with OXOID test discs (OXOID, Basingstoke, Hampshire, England). Routine oral hygiene for the tuboenteral patients was performed by cleansing the oral cavity before meals three times a day with lemon-glycerine wadding sticks impreg- nated with a solution of glycerine-citric acid, lemon flavor- ing, and sodium benzoate 0.1% (7). NGTs in use in our hospital are made from polyvinyl chloride (Duodenal Levin Tube–Maersk Medical, Lynge, Denmark). For the biofilm study, samples of the oropharyngeal section of the NGT were fixed overnight with 2.5% glu- taraldehyde in 0.1 M cacodylate buffer, pH 7.4, washed with the same buffer, dehydrated in increasing concentra- tions of ethanol, dried with a critical point drier, and coat- ed with gold (Polaron-sem coating unit E5100, Thermo VG Scientific, Beverly, MA). The outer surface of the samples was examined by a Jeol-840A scanning electron microscope (JEOL USA, Peabody, MA). Biofilm studies were performed on four patients with an oropharyngeal culture that was positive for P. aeruginosa 2–4 weeks after the NGT was inserted. From three NGT-fed patients with isolations of P. aeruginosa, samples were taken concomitantly from the oropharynx and the NGT surface for strain typing by pulsed-field gel electrophoresis (PFGE). DNA preparation and cleavage with 20 U of Spel endonuclease (New England Biolabs, Eldan, Rosh Ha’ain, Israel) were per- formed, as originally described (8). Electrophoresis was performed in a 1% agarose gel (BMA Products, Hann Woong Yoo, South Korea) prepared and run in 0.5 x Tris- borate-EDTA buffer on a CHEF-DR III apparatus (Bio-Rad Laboratories Ltd. Rishon Le Zion, Israel). The initial switch time was 0.5 s, the final switch time was 35 s, and the run time was 22 h at 6 V/cm with a temperature of 14°C. Gels were stained in ethidium bromide, de-stained in distilled water, and photographed with a Bio-Rad GelDoc 2000 camera. PFGE DNA patterns were compared and interpreted according to the criteria of Tenover et al. (9). The antibiotic susceptibility of P. aeruginosa isolates from the oropharynx was compared to that of P. aerugi- nosa isolates obtained from sputum cultures and recorded in the laboratory logbook in the preceding year. Statistical processing was performed by using SPSS software (SPSS Inc., Chicago, IL). Chi-square test was used for compara- RESEARCH tive studies; p<0.05 was considered significant. Results The study group consisted of 53 elderly long-term-care residents who had been receiving NGT feeding for an average of 14 ± 17 months. The control group consisted of 50 counterparts receiving oral feeding. No statistically sig- nificant differences in demographic and medical character- istics occurred between the study groups (Table). Gram-negative bacteria, including P. aeruginosa, were cultured from the oropharynges of 34 (64%) of the 53 NGT-fed patients. P. aeruginosa was isolated in 18 (34%) patients of this group or in 60% of the patients colonized with gram-negative bacteria. In the control group, gram- negative bacteria were isolated in only 4 (8%) patients (p<0.001). P. aeruginosa was not isolated in any of the control group samples (p<0.001). Two patients from the NGT-fed group had a mixture of other gram-negative bac- teria and P. aeruginosa. No association was found between predisposing factors such as age, gender, diabetes mellitus, chronic lung disease, presence of residual teeth, and isola- tion of P. aeruginosa. Antibiotic susceptibility studies of P. aeruginosa isolat- ed from the oropharynx showed that the highest suscepti- bility rates were registered for tazobactam-piperacillin, with 89% of the isolates being susceptible, followed by ceftazidim (79%) and imipenem, (78%). The P. aerugi- nosa isolates from the oropharynx were less sensitive to most antibiotics than those cultured from sputum; for amikacin, this difference was significant (p<0.03). Figure 1 shows the NGT tube embedded in the adjacent anatomical structures. The findings on electron microscopy of an NGT section are shown in Figure 2. The samples were taken from four patients with cul- tures positive for P. aeruginosa, who had NGTs inserted 2–4 weeks previously. A biofilm with a bacterial organism with a typical form for P. aeruginosa was clearly visual- ized on the outer surface of all four sections. The identity of the bacterium seen in the biofilm was confirmed by cul- ture. Figure 3 shows the results of the PFGE studies on three patients; in each one, the same clone was isolated from both the oral cavity and the NGT. Table. Demographic data of study groupsa NGT-fed patientsa Orally fed patients Data n=53 (%) n=50 (%) Age 78 ± 9 81 ± 9 Dementia 32 (60) 29 (58) Stroke 29 (55) 22 (44) Diabetes mellitus 9 (17) 7 (13) COPD 8 (15) 5 (9) Residual teeth 20 (38) 17 (33) Corticosteroids 6 (11) 4 (8) aNGT, nasogastric tube; COPD, chronic obstructive pulmonary disease. Emerging Infectious Diseases • Vol. 9, No. 8, August 2003 957