Plasmids encoding resistance to aminoglycoside antibiotics have recently been found to be transferred among staphylococci by a mechanism similar to conjugation, a process not previously described in this bacterial genus. Plasmid genes may be essential for their own transfer. Conjugative transfer of aminoglycoside resistance can also mobilize or co-transfer resistance to other antibiotics and may be important in the construction of the multiply-antibiotic-resistance phenotype. Multiresistant hospital-associated staphylococci are often refractory to antibiotic therapy and are the cause of serious outbreaks of nosocomial infections. We propose to investigate the genetic basis of conjugative plasmid transfer among coagulase positive and coagulase negative staphylococci by pursuing the following lines of investigation. First, we will genetically define the transfer or tra region of conjugative plasmids by deletion and transposon insertional mutagenesis and by cloning and subcloning restriction fragments comprising the tra region. DNA will be cloned in E. coli and shuttled to S. aureus by combining plasmids resident in both bacterial hosts. Trans-complementation of cloned fragments will further define tra genes. The radiolabeled tra region will also be used as a probe to identify homologous regions of the same and different species. Second, gene products of the cloned gene will be identified in the E. coli host. Their expression as surface structures on staphylococci will be assessed by raising antibody to tra-plasmid-containing staphylococci and then by using this antibody as a probe to identify any gene products which are surface-expressed. Finally, attempts will be made to interrupt conjugal mating by treating donor or recipient cells with antibody to identified conjugal surface structures. We hope that by genetically defining conjugative antibiotic resistance transfer in staphylococci we can understand the rapid emergence of isolates of this genus that are resistant to multiple antibiotics. These studies will also produce data which may reveal strategies for interrupting transfer and thus halting the spread of nosocomial staphylococcal infections.
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