Multi-clonal evolution of MDR/XDR M. tuberculosis in a high prevalence setting in Papua New Guinea over three decades

20 An outbreak of multi-drug resistant tuberculosis has been reported on Daru Island, Papua New 21 Guinea. The Mycobacterium tuberculosis strains driving this outbreak and the temporal accrual 22 of drug resistance mutations have not been described. We analyzed 100 isolates using whole 23 genome sequencing and found 95 belonged to a single modern Beijing strain cluster. Molecular 24 dating suggested acquisition of streptomycin and isoniazid resistance in the 1960s, with 25 virulence potentially enhanced by a mycP1 mutation. The outbreak cluster demonstrated a high 26 degree of co-resistance between isoniazid and ethionamide (80/95; 84.2%) attributed to an inhA 27 promoter mutation combined with inhA and ndh coding mutations. Multidrug resistance (MDR), 28 observed in 78/95 samples, emerged with the acquisition of a typical rpoB mutation together 29 with a compensatory rpoC mutation in the 1980s. There was independent acquisition of 30 fluoroquinolone and aminoglycoside resistance; with evidence of local transmission of 31 extensively-drug resistant (XDR) strains from 2009. These findings underscore the importance of 32 whole-genome sequencing in informing an effective public health response to MDR/XDR M. 33