We describe a 4.5 kilobase transposon, Tn4001, which mediates resistance to gentamicin, tobramycin and kanamycin in Staphylococcus aureus. Originally detected in plasmid pSK1, Tn4001 was shown to undergo rec-independent transposition to the chromosome from this plasmid and from an inserted derivative of the plasmid pII147. Heteroduplexes between plasmids with and without Tn4001 demonstrated a characteristic stem and loop structure with inverted repeats of approx. 1.3 kilobases.

Nosocomial infections caused by Staphylococcus aureus strains resistant to methicillin and multiple antibiotics have reached epidemic proportions in Melbourne, Australia, over the past 5 years. Plasmid analysis of representative clinical isolates demonstrated the presence of three classes of plasmid DNA in most strains. Resistance to gentamicin, kanamycin, and tobramycin was usually mediated by an 18-megadalton plasmid but could also be encoded by a related 22-megadalton plasmid. Two distinguishable plasmids of 3 megadaltons each endowed resistance to chloramphenicol, and the third class consisted of small plasmids, each approximately 1 megadalton in size, with no attributable function. An extensive array of resistance determinants, including some which have usually been associated with a plasmid locus, were found to exist on the chromosome. Evidence that resistance to gentamicin, kanamycin, and tobramycin is chromosomally encoded in some clinical isolates suggests that this determinant may have undergone genetic translocation onto the staphylococcal chromosome.

All activated sludge systems for removing phosphate microbiologically are configured so the biomass is cycled continuously through alternating anaerobic and aerobic zones. This paper describes a novel aerobic process capable of decreasing the amount of phosphate from 10 to 12 mg P liter ؊1 to less than 0.1 mg P liter ؊1(when expressed as phosphorus) over an extended period from two wastewaters with low chemical oxygen demand. One wastewater was synthetic, and the other was a clarified effluent from a conventional activated sludge system. Unlike anaerobic/aerobic enhanced biological phosphate removal (EBPR) processes where the organic substrates and the phosphate are supplied simultaneously to the biomass under anaerobic conditions, in this aerobic process, the addition of acetate, which begins the feed stage, is temporally separated from the addition of phosphate, which begins the famine stage. Conditions for establishing this process in a sequencing batch reactor are detailed, together with a description of the changes in poly-␤-hydroxyalkanoate (PHA) and poly(P) levels in the biomass occurring under the feed and famine regimes, which closely resemble those reported in anaerobic/aerobic EBPR processes. Profiles obtained with denaturing gradient gel electrophoresis were very similar for communities fed both wastewaters, and once established, these communities remained stable over prolonged periods of time. 16S rRNA-based clone libraries generated from the two communities were also very similar. Fluorescence in situ hybridization (FISH)/microautoradiography and histochemical staining revealed that "Candidatus Accumulibacter phosphatis" bacteria were the dominant poly(P)-accumulating organisms (PAO) in both communities, with the phenotype expected for PAO. FISH also identified large numbers of betaproteobacterial Dechloromonas and alphaproteobacterial tetrad-forming organisms related to Defluviicoccus in both communities, but while these organisms assimilated acetate and contained intracellular PHA during the feed stages, they never accumulated poly(P) during the cycles, consistent with the phenotype of glycogen-accumulating organisms.High levels of phosphate in effluents from activated sludge systems not designed to remove it can lead to toxic cyanobacterial blooms in receiving bodies of water. Consequently, efforts have been directed towards removing phosphate during treatment by microbiological means with a process called enhanced biological phosphorus removal (EBPR), where phosphate is removed from the wasted biomass as intracellular poly(P) (5, 37, 45). Such treatment processes are based on the underlying principle that the biomass needs to be recycled repeatedly through alternating anaerobic and aerobic stages (37), a requirement regarded as crucial for successful EBPR operation. Only after repeated recycling are poly(P)-accumulating organisms (PAO) thought to have a selective advantage over other populations, eventually allowing them to become dominant (5,37,45). In the anaerobic (feed) stage, PAO are belie...

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The Staphylococcus aureus plasmid pSKI carries Tn4001, a 4.7-kilobase (kb) transposon which specifies resistance to gentamicin, tobramycin, and kanamycin. In addition, pSKl mediates resistance to trimethoprim and linked resistance to ethidium bromide (Ebr) and to quaternary ammonium compounds (Qar). Restriction endonuclease analysis of pSK1 and a deleted derivative of pSK1 revealed that the gene(s) responsible for Ebr Qar lies within a 5.2-kb Hindlll fragment. This fragment has been cloned into the Escherichia coli plasmid vector pBR322, and transformants of an E. coli K-12 strain exhibited Ebr Qar. Subcloning of the 5.2-kb insert, combined with data from electron microscopic analysis of deleted derivatives of pSK1, located the Ebr Qar determinant(s) on a 2.3-kb segment of pSK1 DNA.