Chromosomally and extrachromosomally mediated high-level gentamicin

27 Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of sepsis in 28 neonates. The rate of invasive GBS disease in non-pregnant adults also continues to climb. 29 Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin 30 has been shown in vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, 31 leads to loss of a synergistic effect. We therefore performed a multicentre study to determine 32 the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to 33 gentamicin resistance. From eight centres in four countries, 1128 invasive and colonizing 34 GBS isolates were pooled and investigated for the presence of HLGR. We identified two 35 strains that displayed HLGR (BSU1203 and BSU452), both of which carried the aacA-aphD 36 gene, typically conferring HLGR. Though, only one strain (BSU1203) also carried the 37 previously described chromosomal gentamicin resistance transposon, designated Tn3706. In 38 the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in 39 the detection of plasmid pIP501 carrying a remnant of a Tn3 family transposon. Its ability to 40 confer HLGR was proven by transfer into an Enterococcus faecalis isolate. Conversely, loss 41 of HLGR was documented after curing both GBS BSU452 and the transformed E. faecalis 42 strain from the plasmid. This is the first report showing a plasmid mediated HLGR in GBS. 43 Thus, in our clinical GBS isolates HLGR is mediated both chromosomally and 44 extrachromosomally. 45 46