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A prevalence study was carried out on a 100-bed Veterans Administration nursing home care unit to determine the extent of colonization with gentamicin-resistant gram-negative bacilli (GRGNB). Hand cultures of 12 employees and 17 environmental cultures were negative. Twenty-six of 86 (30%) patients were colonized with 49 GRGNB. Sixteen patients (19%) had urinary colonization. Multivariate analysis revealed significant associations between rectal or perineal colonization (P less than 0.01), and the presence of a urinary device (82% condom catheters) (P less than 0.05), with urinary colonization. The most common isolates were Providencia stuartii (20), Escherichia coli (nine) and Klebsiella pneumoniae (nine). Twenty-six of 49 isolates carried plasmids. Restriction endonuclease digestion of plasmid DNA was performed for 21. Cross-colonization, as defined by the presence of the identical species with the identical restriction endonuclease digestion profile of purified plasmid DNA found in different patients, was observed for eight of 21 (38%) strains. All were geographically clustered. No strains could transfer gentamicin-resistance by conjugation and only two plasmids could transform our E coli recipient to gentamicin resistance. One E coli plasmid was identical to two Citrobacter freundii plasmids and a P stuartii plasmid isolated from three different patients. This 105 kb plasmid is conjugative and encodes resistance to ampicillin, carbenicillin, tetracycline, and sulfonamides. Thus, 57% of strains were cross-colonizing or contained identical R-plasmids. Southern hybridization using a 1 kb TEM-1 gene probe demonstrated sequences homologous to this probe in five of five nursing home plasmids examined.(ABSTRACT TRUNCATED AT 250 WORDS)